Analysis of Salivary Metabolites by Nuclear Magnetic Resonance Before and After Oral Mucosa Cleaning of Infants in the Pre-dental Period

Abstract

The aim of the present study was to verify if a protocol for cleaning the oral cavity of infants in the pre-dental period can reduce extrinsic salivary metabolites observed through Nuclear Magnetic Resonance (NMR). A cross-sectional clinical study with a convenience sample was conducted, and infants were recruited at the UFRJ Pediatric Dentistry Clinic. Participants who had used antibiotics and/or antifungals up to 3 months before and whose legal guardians did not consent or sign the Informed Consent Form were excluded. An anamnesis was performed with the guardians and the participants' intraoral clinical examination. Initial collection of unstimulated total saliva was performed using an automatic pipette with sterile plastic tips in the buccal floor region, at least 1 h after the last feeding. Subsequently, the infants' oral mucosa was cleaned with gauze moistened with filtered water, and after 5 min, a new collection was performed, using the same methodology. The obtained samples were immediately transferred on ice to the laboratory, centrifuged (10,000 g), and stored at −80°C. The NMR analyses were performed using a 500-MHz spectrometer Bruker, Germany); evaluations were done via the 1H and 1H-1H TOCSY spectra for metabolite signaling. Eleven pre-dental infants were evaluated, with a mean age of 3.8 months, including six girls (55%). Of these, nine participants (82%) were exclusively breastfed. The higher presence of components such as lactose, glucose, sugars, acetate, alanine, and lactate were observed in the samples before oral mucosa cleaning. Regarding the type of diet, more lactose was observed in the saliva of patients who were exclusively breastfed than those that received mixed feeding. We conclude that the oral mucosa cleaning of infants in the pre-dental period tends to reduce the concentration of extrinsic components from the diet, such as lactose, in the salivary metabolomic profile analyzed by NMR.

Updates and Original Case Studies Focused on the NMR-Linked Metabolomics Analysis of Human Oral Fluids Part III: Implementations for the Diagnosis of Non-Cancerous Disorders, Both Oral and Systemic

“…In 2021, Letieri et al [ 16 ] conducted a cross-sectional clinical investigation coupled with a convenience sample in order to assess the influence of oral mucosa cleansing on the salivary metabolic profiles of infants in their pre-dental periods (mean age 3.8 months); these profiles were acquired through 1 H NMR analysis on a 500 MHz spectrometer. Perhaps unsurprisingly, these researchers found that this oral cleansing approach, which involved the application of gauze moistened with filtered water, resulted in a decrease in glucose, lactose, further sugars, acetate, alanine and lactate, which may all arise from dietary sources anyway.…”

NMR-Based Metabolomics Demonstrates a Metabolic Change during Early Developmental Stages from Healthy Infants to Young Children

“…A mixture of 540 μL of saliva supernatant from the respective subjects plus 60 μL of pH 7 phosphate buffer was prepared, containing 99.8% (to provide a field-frequency lock) and 20 µM of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) (for chemical shift referencing, δ = 0.00 ppm) [ 14 , 29 ]. The spectra acquisitions (0–12 ppm) were performed in a standardized way, with a pre-established receiver gain and number of scans (1024 scans) and a calculated pulse time and signal/noise ratio.…”

“…Data were subjected to correction and base alignment using the software Topspin 4.1.4 (Bruker Biospin program, Rheinstetten, Germany). 1 H- 1 H total correlation (TOCSY) experiments were also conducted, with acquisition parameters of 2048 × 256 complex points, a spectral width of 12,019 Hz in each dimension, and a mixing time of 70 ms [ 8 , 14 ]. Spectra and spectral regions that could not be corrected for phase and baseline were excluded from the analyses.…”

“…Spectra and spectral regions that could not be corrected for phase and baseline were excluded from the analyses. The marking strategy included the use of the Human Metabolome database ( , accessed on 24 June 2022 ) and previous markings in the literature [ 8 , 14 , 29 ]. TOCSY experiments were used to confirm the assignments.…”

“…[ 10 , 11 , 12 , 13 ]. To this end, this group characterized the profile of salivary metabolites in healthy children of different ages, including infants [ 14 ], with and without caries [ 8 , 15 ]. This study also determined the metabolites that are produced by oral microbiota metabolism [ 9 , 16 ] which characterize systemic diseases in children, as well as type I diabetes [ 12 ] and renal failure [ 11 , 13 ].…”