¹H NMR Investigations of Diurnal Metabolic Variations in Human Saliva

High-resolution ¹H nuclear magnetic resonance (NMR) analysis serves as a powerful means of probing the metabolic status of human saliva. Objectives: Since the excretion of selected metabolites in this biofluid serve as 'markers' of microbicidal activity and soft-tissue inflammation, we employed this technique to track diurnal modifications in the salivary composition of a group of subjects using a pre-allocated dentifrice. Methods: Non-medically-compromised subjects (n = 10) were provided with a NaF-containing toothpaste [1] and were permitted to brush as usual (2 x daily) for 2 weeks in order to establish 'baseline' salivary metabolic data. During weeks 3 and 4, each subject supplied saliva specimens at 5 daily time-points (0, 30, 120, 360 and 600 min. post-brushing) on 3 separate days. The time 0 (pre-brush) sample was collected immediately following a.m. 'wake-up' and prior to performance of oral activities. ¹H NMR measurements were made on a Bruker Avance AX-600 spectrometer. Statistical analysis of salivary concentration data was conducted by ANOVA. Results: Significant time-dependent trends were observed in the concentrations of selected salivary biomolecules: i.e. lactate and acetate serving as markers of caries, propionate, n- and iso-butyrates as indicators of periodontal disease, and malodorous di- and trimethylamines associated with halitosis. Indeed, all of these metabolites had elevated salivary levels in the a.m. pre-brushing samples (p << 0.05) and, with the exception of lactate, significantly lower and relatively constant concentrations at each subsequent diurnal time-point (plots of lactate level versus time-point revealed diurnal 'spikes'). Conclusions: Multicomponent ¹H NMR analysis of salivary biomolecules provides much valuable information regarding the diurnal variation of metabolic markers of oral disease activity. Results acquired revealed that overnight periods represent a significant challenge to oral health (despite previous night-time hygeine measures), and therefore products specifically designed for long-term, overnight action may provide unique oral healthcare advantages. [1] Crest Decay Prevention toothpaste, The Procter and Gamble Company, USA.