## 2 Sources and Composition of Human Saliva: Host and Microbial Contributions
### 3.1. Ease of Collection and Metabolite Concentration Considerations  
As with blood plasma and urine, saliva is a highly complex biofluid containing a multitude of biomolecules that are receptive to \({}^{1}\)H NMR analysis, along with other analytical strategies for this spectroscopic technique such as homo- and heteronuclear 2D techniques. However, like plasma, but unlike urine, saliva has significant levels of proteins present [18, but, as noted above, this is only approximately \(\leq\)10% or so of that of blood plasma, and we find that this offers little or no interference with the \({}^{1}\)H NMR determination of salivary metabolites [2; 3; 4]; even healthy human urine contains trace levels of proteins, which in the majority of cases do not obstruct the quantitative analysis of biomolecules present in this biofluid. Similarly, all these biofluids also contain a broad'spectrum' of non-NMR-detectable or -visible biomolecules; the latter analytes are below pre-specified LLOD and hence lower limits of quantification (LLOQ) limits for these agents. Although the conventional and traditional analyses of blood or blood plasma/serum samples is considered integral for the majority of clinical diagnostic and prognostic monitoring work-tasks, methods or protocols available for their collection are hampered by its high expense (most especially in health services), its invasive physical intrusion, the requirement for a trained healthcare specialist (e.g., phlebotomist or trained nurse) for this purpose,and the possibility of adverse reactions to the collection technique experienced by a very small but nevertheless significant proportion of study participants (on Research Ethics Committee-approved participant information sheets, it is usual to warn participants about these possible adverse side-effect associated with blood sample collection, e.g., experiencing dizziness or feeling faint, etc.). However, the collection of saliva is virtually non-invasive and non-intrusive, and can be achieved by patients/participants themselves, often in their own homes, without any clinical supervision required. This non-invasive collection strategy for the detection and/or monitoring of clinical conditions clearly circumvents the discomfort often associated with blood sample collection. In addition to the simple straightforward, non-invasive, and painless method of saliva sample aspiration (albeit subject to a number of important requirements), further major advantages associated with the use of saliva as a sample matrix in NMR-linked metabolomics studies are that (1) samples are more readily shipped and stored--saliva requires much less laboratory handling and processing than whole blood, and it obviously does not clot; (2) its collection is economical, which reduces costs for healthcare providers and/or researchers; and (3) samples are safer to handle--notably, saliva naturally contains agents that suppress HIV infectivity, and therefore its handling and laboratory processing presents risks of only extremely restricted or negligible rates of oral HIV transmission [39]. Notwithstanding, one already established major disadvantage of the use of saliva in a metabolomics and general biomarker analysis context for extra-oral diseases is the knowledge that levels of such monitorable analytes are always lower or substantially lower in this biofluid than they are in blood samples, as whole, or its plasma/serum isolates [40]. For example, the high-molecular-mass marker IgG has a blood serum concentration lying between 5-30 g/L but is 1000-fold lower in corresponding saliva specimens [41]. Furthermore, for IgA, serum levels are 2.5-5.0 g/L, whereas they are only 0.25-0.50 g/L for saliva [41], i.e., a 10-fold lower mean difference.