Metabolic profiling by1H-NMR spectroscopy is an under-utilised technology in salivary research although preliminary studies have identified promising results in multiple fields (diagnostics, nutrition, sports physiology). Translation of preliminary findings into validated, clinically approved knowledge is hindered by variability in protocol for the collection, storage, preparation and analysis of saliva. This study aims to evaluate the effects of differing sample pre-treatments on the1H-NMR metabolic profile of saliva. Protocol considerations are highly varied in the current literature base, including centrifugation, freeze-thaw cycles and different NMR quantification methods. Our findings suggest that the1H-NMR metabolite profile of saliva is resilient to any change resulting from freezing, including freezing of saliva prior to centrifuging. However, centrifugation was necessary to remove an unidentified broad peak between 1.24 and 1.3 ppm, the intensity of which correlated strongly with saliva cellular content. This peak obscured the methyl peak from lactate and significantly affected quantification. Metabolite quantification was similar for saliva centrifuged between 750 g to 15,000 g. Quantification of predominant salivary metabolites (acetate, lactate and propionate) were similar whether quantified using internal phosphate-buffered sodium trimethylsilyl-[2,2,3,3-2H4]-propionate (TSP) or external TSP in a co-axial NMR tube placed inside the NMR tube containing the saliva sample. Our results suggest the existing literature on salivary1H-NMR will not have been adversely affected by variations of the common protocol, however, use of TSP as an internal standard without a buffered medium appears to affect metabolite quantification. We include protocol recommendations to facilitate future NMR-based studies of saliva.
- Journal Article