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

Abstract

Metabolic profiling by H 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 theH 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 H 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 750 g to 15 000 g. Quantification of salivary metabolites was similar whether quantified using internal phosphate-buffered sodium trimethylsilyl-[2,2,3,3-H]-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 H 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.

Determining bacterial and host contributions to the human salivary metabolome

“…Saliva collection is less invasive than plasma collection, and saliva can be produced adlibitum unlike urine [1]. To allow the full diagnostic potential of saliva to be reached, much research pertaining to salivary biomarker discovery currently emphasises optimisation either of sample handling or analytic techniques [2][3][4][5][6] and assessing physiological modifiers in health such as diurnal variation, diet and age [7,8].…”

Current Status of Magnetic Resonance on Saliva for Oral and Dental Health Early Diagnosis

“…The standardization of the methodology is of high importance. The significant inter-study variability has been pointed out regarding saliva sample storage, preparation, and NMR quantification method, in contrast to the established methodology for other biofluids (e.g., plasma, urine) [19]. For the clinical application of saliva testing as a diagnostic method, specific sampling collection indications and guidelines need to be established as well, which should be based on the specific health condition that is under investigation.…”

“…NMR, as a powerful analytical method, can identify and quantitively analyze organic acids, amino acids, fatty acids, amines, and alcohol in the provided biofluid sample. Until now, it has been mainly employed for the analysis of plasma and urine [19].…”

“…Notably, the general NMR inherent insensitivity compared with mass spectroscopy can be improved using multiple scans, cryoprobes, and higher magnetic fields [22,23]. Regarding the current status of NMR in saliva metabolite analysis, it has been found to be underutilized compared with its potential, and a standardized protocol needs to be followed for its saliva analysis application [17,19].…”

Hochdurchsatz‐Metabolomik mit 1D‐NMR

“…So lassen sich die kritischen Schritte und Parameter identifizieren, die die Konzentration der Metabolite beeinflussen, die schnellem Abbau unterliegen (mit der Folge, dass sich die Abbauprodukte anhäufen). Für einige der häufigsten Proben gibt es systematische Untersuchungen; dabei wurden enzymatische Aktivitäten in den Proben als wichtigste Schwankungsquelle entdeckt. Stammen die Proben aus Blut und verwandten Flüssigkeiten, beeinflussen Zellen in der Probe stark die Konzentration von Glucose und Lactat; die Gewinnung von Serum und Plasma sollte daher spätestens 30 min nach der Blutentnahme begonnen werden.…”