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

Aline Dos Santos Letieri,Ivete Pomarico Ribeiro De Souza,Tatiana Kelly Da Silva Fidalgo,Ana Paula Valente,Gabriela Pereira Fontes,Liana Bastos Freitas-Fernandes,Lourenço Luís Albarello

doi:10.3389/fdmed.2021.667365

Aline Dos Santos Letieri, Ivete Pomarico Ribeiro De Souza + Show 5 more

Open Access

https://doi.org/10.3389/fdmed.2021.667365

Copy DOI

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.

Highlights

Saliva is a highly complex biofluid, produced by the secretion of the major and minor salivary glands, the gingival crevicular fluid, residual food, and microorganisms and their metabolic products, presenting a wide spectrum of biological compounds with informative capacity
These findings are extremely important for the determination of an efficient, reproducible, and accurate salivary collection protocol for infants to avoid interference of milk peaks and other extrinsic components from the diet directly interfere with the Nuclear Magnetic Resonance (NMR) analysis of the salivary metabolites
It has previously been shown that performing saliva collections in infants without previous cleaning of their mouths directly interfered with the metabolomics analysis through NMR, especially because it presents extremely intense peaks in the sugar region, acting as a confounding factor and making it difficult to obtain accurate and reliable results for the other salivary metabolites [18]

Summary

Introduction

Saliva is a highly complex biofluid, produced by the secretion of the major and minor salivary glands, the gingival crevicular fluid, residual food, and microorganisms and their metabolic products, presenting a wide spectrum of biological compounds with informative capacity. The metabolites are classified as intrinsic when they are produced by the cellular metabolism of body itself or extrinsic, which are substances foreign to biological samples, such as drugs or products resulting from the microorganisms’ metabolism [3]. Researches on the evaluation of salivary samples are of fundamental relevance, since saliva is an important and promising vehicle for early diagnosis, preventive use, and even for the identification of biomarkers for local and systemic diseases, mainly due the advantages of presenting simple and non-invasive forms of collection and because of sufficient precision when compared to traditional methods [4, 5]

Objectives

Methods

Results

Conclusion