Molecular Identification of Salivary Metabolites in Children with Early Childhood Caries

Background: Early childhood caries has been a continuous threat to overall quality of life of children, largely irrespective of their different socio-economic status and other factors. In spite of quality researches being done all over world, many questions related to this disease remain unanswered. The present study is an attempt to explore few relatively lesser explored areas of ECC.

Methods: Children within 3-5 years of age group were divided into ECC (n=40) and Caries free (n=33) groups. Their clinical assessment was done, demographic data were recorded and saliva samples were collected and transported to concerned laboratory with standardized protocol. Saliva samples were frozen at -80 °C until NMR analysis was performed. NMR experiments were conducted on Bruker Avance II 400 MHz spectrometer equipped with a BBI probe (Bruker Biospin, Germany). One-dimensional 1H NMR measurements were obtained using Carr–Purcell–Meiboom–Gill (CPMG) sequence with water suppression by pre-saturation and noesypr1D sequence at 300 K. To aid spectral assignments, two dimensional Homonuclear (TOCSY) and Heteronuclear (HSQC) NMR experiments were used. Spectral data were further subjected to multivariate analysis.

Results: Proton (1H) NMR spectra obtained from healthy and ECC subjects were processed and utilized for characterization of metabolites. Characterization of these metabolites was carried out on the basis of chemical shift, coupling constant and splitting pattern of metabolites as reported in literature. Till now 15 metabolites were indentified, which includes Formate, Lactate, Tyrosine, Histidine, Acetate, Alanine, Propionate, Butyrate, Phenylalanine, Glycine, Taurine, Dimethylamine etc. and further assignment, quantitation and multivariate data analysis is in progress.

Conclusion: NMR spectroscopy is a powerful and useful tool for identifying the various metabolites present in saliva, which in turn may prove useful in biomarker discovery related to ECC.