Abstract
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.
Highlights
Metabolomics focuses on the high-throughput measurement of low-molecular-weight compounds comprised in a biological system [1,2]
The spectrum exhibits a large number of narrow peaks, arising from low molecular weight (Mw ) compounds (e.g., many amino acids, short chain fatty acids (SCFA), organic acids, carbohydrates and others, Table S1), often superimposed on broader resonances, which arise from high Mw compounds
One of the aims of this study was to evaluate the stability of saliva samples temporarily stored at 22 ◦ C, 4 ◦ C, or −20 ◦ C, before they can be transferred to the ideal conditions of long-term storage at −80 ◦ C
Summary
Metabolomics focuses on the high-throughput measurement of low-molecular-weight compounds comprised in a biological system [1,2]. Saliva metabolomics has been relatively underexplored, compared to blood or urine, growing interest has been noted in the last decade [7,8], to find biomarkers of oral pathologies [9,10,11,12], and of several other diseases, such as T1DM [13] and dementia [14,15]. Easy and non-invasive saliva collection adds to its interest in disease research, as it enhances the compliance of elderly and children. It is important, to establish specific standard operating procedures (SOPs) to ensure saliva integrity throughout all steps of the metabolomics process
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