## 8 Homo- and Heteronuclear Two-Dimensional (2D) NMR Analysis of Human Saliva
### Sample Size Considerations and MV Power Calculations
The generation of plaque ammonia proceeds via urea hydrolysis by the enzyme urease or from arginine through arginine deaminase, systems that are viewed to act protectively against the development of dental caries. The cariogenic species _Streptococcus mutans_[152] has an ammonia-producing agmatine deaminase complex that degrades agmatine to putrescine, ammonia and CO\({}_{2}\) and may represent a suitable target system for anti-caries therapeutic approaches. Moreover,as we might expect, caries-free individuals have been found to display a higher urease- and arginine-deiminase system-induced ammonia generating activity, in both saliva and plaque samples, than those with low caries vigor [153]. Therefore, caries-free individuals appear to have relatively greater levels of ammonia generation in their oral environments. Figure 10: Two-dimensional correlation heatmap of the 31 \({}^{1}\)H NMR-assigned metabolites employed for factor analysis of orthogonal salivary metabolite pools in a cohort of 48 healthy control participants (five daily saliva samples were collected from each subject). The top and left-hand side ordinate axes show the application of a correlation-based agglomerative hierarchical clustering analysis of the full dataset. Abbreviations: His, Histidine; Form, Formate; Val, valine; MA, DMA and TMA, Methyl-, Dimethyl- and Trimethylamine respectively; iso-But, _iso_-Butyrate; MeOH, methanol; N-Ac-1, -2 and -3, N-Acetyl storage compound resonances located at \(\delta\) = 2.025/2.030, 2.040 and 2.053/2.058 ppm respectively (Table 1); Glu, Glutamate; Gln, Glutamine; Suc, Succinate; Pyr, Pyrruvate; Chol, Choline; Gly, glycine; Lac, Lactate; Tau, Taurine; Phe, Phenylalanine; Tyr, Tyrosine; Ace, Acetoin; Thy, Thymine; 5-AV, 5-Aminovalerate; 3-D-HB, 3-D-Hydroxybutyrate; Ala, Alanine; Prop, Propionate; A, Acetate; n-Cap, \(n\)-Caproate; \(n\)-But, \(n\)-Butyrate. _iso_-Butyrate loaded strongly on PC4, and [PERSON] et al. [154] observed that this organic acid anion originated from the bacterial catabolism of a valine substrate in human stool, and along with iso-valerate, it may indicate fundamental protein maldigestion or malabsorption; although this amino acid was found to predominantly load on PC3 arising from the above factor analysis of our salivary metabolite dataset, it secondarily also loaded on PC4, although with a loading vector of only 0.30 (Table 5). However, this at least indicates some correlation between them. Also loading on PC4 is a combination of histidine and formate, which would be expected to be associated in view of the former serving as a metabolic precursor of the latter [155]. However, serine, glycine, methionine, choline, and methanol can also be processed by selected endogenous mammalian metabolic systems to produce formate [156]. Formate in the oral environment remains of some significance in view of its crucial involvement in one-carbon metabolic biochemistry in vivo [156], as a potential marker of toxicity from the metabolism of salivary methanol via formaldehyde [58], and its markedly elevated salivary levels (range 0.20-61 mmol./L) [3] over those of other biofluids such as blood plasma.