Evaluation of Saliva Stability for NMR Metabolomics: Collection and Handling Protocols.

Maintaining a salivary metabolic profile upon sample collection and preparation is determinant in metabolomics. Nuclear magnetic resonance (NMR) spectroscopy was used to identify metabolite changes during short-term storage, at room temperature (RT)/4 °C/-20 °C, and after sample preparation, at RT/4 °C (mimicking typical clinical/laboratory settings). Interestingly, significant metabolic inter-individual and inter-day variability were noted, probably determining sample stability to some extent. After collection, no changes were noted at -20 °C (at least for 4 weeks). RT storage induced decreases in methylated macromolecules (6 h); lactate (8 h); alanine (12 h); galactose, hypoxanthine, pyruvate (24 h); sarcosine, betaine, choline, N-acetyl-glycoproteins (48 h), while acetate increased (48 h). Less, but different, changes were observed at 4 °C, suggesting different oral and microbial status at different temperatures (with a possible contribution from inter-individual and inter-day variability), and identifying galactose, hypoxanthine, and possibly, choline esters, as potential general stability indicators. After preparation, addition of NaN3 did not impact significantly on saliva stabilization, neither at RT nor at 4 °C, although its absence was accompanied by slight increases in fucose (6.5 h) and proline (8 h) at RT, and in xylose (24 h) at 4 °C. The putative metabolic origins of the above variations are discussed, with basis on the salivary microbiome. In summary, after collection, saliva can be stored at RT/4 °C for up to 6 h and at -20 °C for at least 4 weeks. Upon preparation for NMR analysis, samples are highly stable at 25 °C up to 8 h and at 4 °C up to 48 h, with NaN3 addition preventing possible early changes in fucose, proline (6-8 h), and xylose (24 h) levels.