## 8 Homo- and Heteronuclear Two-Dimensional (2D) NMR Analysis of Human Saliva
### Sample Size Considerations and MV Power Calculations
Notwithstanding, _MetSizeR_ is essentially a targeted approach, since the maximum number of variables that it can accommodate is 375, but fortunately this is usually more than sufficient for many bucketed \({}^{1}\)H NMR datasets, unlike the DSD approach that accommodates larger datasets. Standard parameters for this method are specified in [140], and an example featured in this report explored an \({}^{1}\)H NMR-based rat urinary dataset (n = 36) split into two with samples longitudinally collected throughout a 15-day duration. Application of the above two power analysis methods successfully confirmed that \(\leq\)30 samples were required as optimal sample sizes in future datasets acquired on this experimental system. SSPA from the _Bioconductor R_ package also serves as a valuable methodology [141; 142]. Readers are referred to [135] for an outline summary of data processing, modeling and power calculations using both UV and MV analysis strategies available for the evaluation and interpretation of'state-of-the-art' metabolomics datasets, \({}^{1}\)H NMR-based or otherwise. Significance of between-Participant Sources of Metabolite Level Variance in Healthy Humans: Salivary Phenotype Analysis  
In 2016, [PERSON] et al. [30] reported on the abilities of different saliva sample donors to generate characteristic, participant-dependent salivary metabolic signatures. For this purpose, they employed \({}^{1}\)H NMR-based metabolomics analysis to investigate saliva specimens collected from a total of 23 healthy volunteers, with ca. 6 samples donated daily from each of these for a 10 consecutive day duration (seven days under what was described as'real-life' circumstances and three whilst undergoing a standardized diet regimen, together with a physical exercise program at Day 10). Although the authors claim that this was the very first demonstration of nascent individual salivary metabolic phenotypes in humans, the roots of this study can be found in [3], since, in that investigation, our group demonstrated that there was indeed a significant 'between-participant' component of variance (which was tested against a 'within-participant' one) in 9 out of 11 salivary metabolites quantified and monitored. Notwithstanding, in [30], the researchers involved found that this individual salivary phenotype was not quite as strong as that found for urine but was affected less by diet. Hence, a further concern of ours is that we are fully aware that if saliva specimens are collected within an insufficient time following meal consumption (from Figure 2, we recommend an absolute minimum of at least 4 h, but a period of 8 or even 12 h is certainly preferable), the \({}^{1}\)H NMR profiles of human saliva are contaminated with large numbers of exogenous resonances from food-borne agents, particularly carbohydrates, organic acid anions, and lipids, etc. Indeed, if samples are collected shortly after meals, including those donated only 1.0 h thereafter, then it is often impossible or virtually impossible to view many of the expected endogenous metabolite signals in such spectra. Moreover, dietary carbohydrates such as glucose or sucrose immediately trigger the salivary generation of a series of lower molecular mass catabolites, and in the current study we found substantial changes in the levels of endogenous, non-food nutrient-sourced metabolites, notably lactate, pyruvate, and succinate, amongst those of others (Figure 2), which arise from the fermentation of dietary carbohydrates. As expected, oral microbiome responses to fermentable carbohydrate exposure are reflected through modifications of the salivary metabolome, including saccharolytic bacterial-mediated rises in acetate, propionate and \(n\)-butyrate levels, for example. Although known for some time [143], this phenomenon has been monitored via metabolomics techniques in dental plaque [144; 145], and sucrose-induced challenges observed therein were associated with those occurring in saliva.