Updates and Original Case Studies Focused on the NMR-Linked Metabolomics Analysis of Human Oral Fluids Part I: Emerging Platforms and Perspectives

1H NMR-based metabolomics analysis of human saliva, other oral fluids, and/or tissue biopsies serves as a valuable technique for the exploration of metabolic processes, and when associated with ’state-of-the-art’ multivariate (MV) statistical analysis strategies, provides a powerful means of examining the identification of characteristic metabolite patterns, which may serve to differentiate between patients with oral health conditions (e.g., periodontitis, dental caries, and oral cancers) and age-matched heathy controls. This approach may also be employed to explore such discriminatory signatures in the salivary 1H NMR profiles of patients with systemic diseases, and to date, these have included diabetes, Sjörgen’s syndrome, cancers, neurological conditions such as Alzheimer’s disease, and viral infections. However, such investigations are complicated in view of quite a large number of serious inconsistencies between the different studies performed by independent research groups globally; these include differing protocols and routes for saliva sample collection (e.g., stimulated versus unstimulated samples), their timings (particularly the oral activity abstention period involved, which may range from one to 12 h or more), and methods for sample transport, storage, and preparation for NMR analysis, not to mention a very wide variety of demographic variables that may influence salivary metabolite concentrations, notably the age, gender, ethnic origin, salivary flow-rate, lifestyles, diets, and smoking status of participant donors, together with their exposure to any other possible convoluting environmental factors. In view of the explosive increase in reported salivary metabolomics investigations, in this update, we critically review a wide range of critical considerations for the successful performance of such experiments. These include the nature, composite sources, and biomolecular status of human saliva samples; the merits of these samples as media for the screening of disease biomarkers, notably their facile, unsupervised collection; and the different classes of such metabolomics investigations possible. Also encompassed is an account of the history of NMR-based salivary metabolomics; our recommended regimens for the collection, transport, and storage of saliva samples, along with their preparation for NMR analysis; frequently employed pulse sequences for the NMR analysis of these samples; the supreme resonance assignment benefits offered by homo- and heteronuclear two-dimensional NMR techniques; deliberations regarding salivary biomolecule quantification approaches employed for such studies, including the preprocessing and bucketing of multianalyte salivary NMR spectra, and the normalization, transformation, and scaling of datasets therefrom; salivary phenotype analysis, featuring the segregation of a range of different metabolites into ‘pools’ grouped according to their potential physiological sources; and lastly, future prospects afforded by the applications of LF benchtop NMR spectrometers for direct evaluations of the oral or systemic health status of patients at clinical ‘point-of-contact’ sites, e.g., dental surgeries. This commentary is then concluded with appropriate recommendations for the conduct of future salivary metabolomics studies. Also included are two original case studies featuring investigations of (1) the 1H NMR resonance line-widths of selected biomolecules and their possible dependence on biomacromolecular binding equilibria, and (2) the combined univariate (UV) and MV analysis of saliva specimens collected from a large group of healthy control participants in order to potentially delineate the possible origins of biomolecules therein, particularly host- versus oral microbiome-derived sources. In a follow-up publication, Part II of this series, we conduct censorious reviews of reported observations acquired from a diversity of salivary metabolomics investigations performed to evaluate both localized oral and non-oral diseases. Perplexing problems encountered with these again include those arising from sample collection and preparation protocols, along with 1H NMR spectral misassignments