Establishment of a NMR-based metabolomics protocol for salivary samples

In contrast to the most used biofluids such as blood and urine, saliva is rarely studied in metabolomics. However, this biofluid seems to be of great interest because of its non-invasive, simple and fast collection. Moreover, it is perfectly suited for self-sampling, well-adapted to a personalized approach of medicine and is expected to be complementary with other biofluids information. This is why we aim to develop and optimize a protocol to analyse salivary samples by NMR.
Therefore, this work focused on the identification of the best suitable protocol allowing to obtain the most pertinent and huge metabolomics information. For this purpose, four methods were selected according to the literature and to in-house processes and were compared. On the basis of several analytical criteria such as the number and the concentration of identified metabolites, the repeatability and the robustness, the best method was selected. Freeze-drying step followed by ultrafiltration led to the most informative and repeatable protocol. Finally, this optimized workflow was applied to a real-case study (fasting vs non-fasting volunteers) in order to prove the pertinence of the method and of the saliva's analysis in metabolomics. This study showed significant differences between the groups and discriminant metabolites were identified.
In conclusion, results obtained in this work are encouraging and highlight the real interest of using saliva samples in metabolomics. In the future, it will be interesting to apply the developed protocol to metabolomics studies and to combine it with the usual blood and urine's metabolomics analyses.