Developing and Standardizing a Protocol for Quantitative Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy of Saliva

Metabolic profiling by 1H NMR spectroscopy is an underutilized technology in salivary research, although preliminary studies have identified promising results in multiple fields (diagnostics, nutrition, sports physiology). Translation of preliminary findings into validated, clinically approved knowledge is hindered by variability in protocol for the collection, storage, preparation, and analysis of saliva. This study aims to evaluate the effects of differing sample pretreatments on the 1H NMR metabolic profile of saliva. Protocol considerations are highly varied in the current literature base, including centrifugation, freeze−thaw cycles, and different NMR quantification methods. Our findings suggest that the 1H NMR metabolite profile of saliva is resilient to any change resulting from freezing, including freezing of saliva prior to centrifuging. However, centrifugation was necessary to remove an unidentified broad peak between 1.24 and 1.3 ppm, the intensity of which correlated strongly with saliva cellular content. This peak obscured the methyl peak from lactate and significantly affected quantification. Metabolite quantification was similar for saliva centrifuged between 750g to 15 000g. Quantification of salivary metabolites was similar whether quantified using internal phosphate-buffered sodium trimethylsilyl-[2,2,3,3-2H4]-propionate (TSP) or external TSP in a coaxial NMR tube placed inside the NMR tube containing the saliva sample. Our results suggest that the existing literature on salivary 1H NMR will not have been adversely affected by variations of the common protocol; however, use of TSP as an internal standard without a buffered medium appears to affect metabolite quantification, notably for acetate and methanol. We include protocol recommendations to facilitate future NMR-based studies of saliva.

-1: Summary of selected salivary metabolite concentrations collected pre-, during and 2 h post-exercise.

Supplementary Information
Factors affecting salivary metabolites Exogenous substances Figure S-1: (A) Partial 700 MHz CPMG 1 H-NMR spectra (echo time 64 ms, 3.00 -4.10 ppm) of saliva collected from the same participant before and twenty minutes after eating. Spectra are of the same vertical scale. Peaks from sucrose, maltose and glucose obscure metabolites such as glycine and taurine and the quartet from lactate (4.12 ppm) in saliva collected twenty minutes after eating (i). These peaks were not observed in samples collected one hour after eating or drinking (ii). (B) Detection of xylitol from chewing gum in saliva collected one hour after chewing gum. Xylitol peaks did not obscure other assigned metabolites. Samples were centrifuged at 15,000 g prior to freezing.

Intra-oral catabolism of dietary substances
Certain exogenous substances not only obscure salivary NMR spectra but are readily metabolised in the oral cavity by the complex microbial community, and thus alterations in levels of other salivary metabolites can be observed. This is illustrated in Figure S2, showing the intra-oral catabolism of sucrose. Water (10 ml) was held in the mouth for 30 s before being expectorated. Saliva was then collected over a period of two minutes. This process was repeated after 5 mins with 0.25 M sucrose solution (10 ml).
Studies of saliva involving consumption of oral substances (including those administered as sialagogues, e.g. citric acid) therefore need to consider the effects these substances may have on metabolite profile.

Exercise induced changes
Changes in salivary metabolite concentration were induced by exercise. Within ten minutes of continuous exercise, expectorated saliva had higher concentrations of all metabolites Levels returned to baseline within two hours post-exercise. The higher levels during exercise may partly be due to dehydration (i.e. less fluid leading to more concentrated metabolites), however metabolite concentrations do not change proportionally, indicating additional factors causing differential generation and consumption of metabolites. Recent exercise therefore presents an additional variable to consider prior to collecting saliva for 1 H-NMR spectroscopy.

Figure S-3:
Partial 700 MHz CPMG 1 H-NMR spectral regions (0.8 -2.5 ppm) of saliva collected (A) before, (B) during and (C) two hours after exercise. Samples were centrifuged at 15,000 g prior to freezing, with quantification by internal, buffered TSP. Vertical scale is the same for all spectra. The acetate peak has been truncated. Comparison of saliva with CPMG and NOESY pulse sequences Figure S-4: Stacked partial spectra of partial 700 MHz 1 H-NMR spectral regions (0.1 -4.1 ppm) of the same saliva sample analysed with a NOESY pulse sequence (top) and a CPMG pulse sequence (echo time 64 ms, bottom). Spectra are of the same vertical scale with the acetate and lactate peaks truncated. Spectra were similar, however, the CPMG spectra featured a flatter baseline than the NOESY spectrum, without attenuating the remaining resonances, and so the former was used for quantification. NOESY 1 H-NMR spectra were acquired at 700.13 MHz. 32 transients were collected with 64 k data points following four dummy scans, with a spectral width of 20 ppm (-5 to 15 ppm), a relaxation delay of 4 s and a mixing time of 10 ms.

S-5
S-7 Absolute volumes are not important, provided the volume read by the NMR probehead (rectangular area) is fully covered. Once the spectrum has been acquired and the peak integrals measured the volume ratio can be calculated using the equation:

= *
Where proton concentration is the molar concentration of the solution multiplied by number of protons contributing to the peak that was integrated. Diagram is not to scale.

Figure S-6:
Confirmed assignment of acetoin in whole mouth unstimulated saliva by 2D NMR. 2D 1 H-1 H COSY spectra were obtained from saliva samples of two participants, A and B. Spectra were acquired with 4096 data points, 400 increments, with 8 scans per increment, a relaxation delay of 2 s and spectral width 11,160 Hz (15.9 ppm). In both saliva samples, the doublet at 1.37 ppm (arising from the methyl group adjacent to the CH(OH) in acetoin) shows a cross peak at 4.42 ppm, which matches HMDB assignments for acetoin (http://www.hmdb.ca/spectra/nmr_one_d/1939). The quartet at 4.42 ppm from the proton in the CH(OH) group is masked by other resonances in the 1D 1 H-NMR spectra of saliva. Samples were centrifuged at 15,000 g prior to freezing, with quantification via external TSP in a coaxial tube.
S-9 . The corresponding 2D 1 H-1 H COSY spectra shows a faint cross peak at 1.13 and 3.64 ppm, thought to be propylene glycol (C) but on addition of propylene glycol, another cross peak is evident at 1.13 and 3.87 ppm (D), suggesting the 1.13 ppm resonance in saliva does not arise from propylene glycol as previously assigned from the 1D-1 H NMR spectra.. Samples were centrifuged at 15,000 g, and analysed fresh. No standard was used as spectra were calibrated using the acetate peak. Spectra A and B are at the same vertical scale.

S-11
Comparison of different freeze-thaw treatments on the 1 H-NMR spectra of saliva Figure S-9: Stacked partial spectra of partial 700 MHz CPMG 1 H-NMR spectral regions (0.6 -3.6 ppm) of saliva sample collected at the same time from a representative individual. Spectra are of: (i) centrifuged at 15,000 g with no freezing; (ii) frozen and thawed after centrifuging at 15,000 g; (iii) frozen and thawed before centrifuging at 15,000 g; (iv) centrifuged at 15,000 g and then frozen and thawed four times. Quantification was via external TSP in a coaxial tube for all aliquots. The same degree of similarity was observed in the other participants. Spectra are of same vertical scale. The acetate peak has been truncated.