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.