NMR Analysis of Saliva Metabolites

Samples were processed for NMR using a previously published method (Cleaver et al., 2019 (link)). Briefly, centrifuged spent saliva supernatant and the sterile saliva sample were each mixed with TSP buffer (one part 2 mM sodium trimethylsilyl-[2,2,3,3-²H₄]-propionate, 28.4 mg/ml Na₂HPO₄ and 5.28 mg/ml NaH₂PO₄ to two parts 50% by volume deuterium oxide, sample:buffer ratio 4:1) in a 5 mm NMR tube (Bruker, Germany). The tubes were sealed and analyzed at the Biomolecular Spectroscopy Centre, King's College London, UK on a 600 MHz spectrometer (Bruker) for ¹H 1D-NMR and ¹H-C¹³ 1D- and 2D-NMR. The concentration of metabolites were collected using Chenomix NMR Suite version 8.5 (Chenomix Ltd., Canada). The 2D C¹³ spectra were analyzed using TopSpin version 3.6.2 (Bruker) and COLMAR (Bingol et al., 2014 (link)).