Differences in metabolic profile between submandibular, parotid and whole saliva: a new concept in salivary diagnostics.

Event Abstract Back to Event Differences in metabolic profile between submandibular, parotid and whole saliva: a new concept in salivary diagnostics. Rita Antonelli1*, Thelma Pertinhez1, 2, Margherita Eleonora Pezzi1, Benedetta Ghezzi1, Paolo Vescovi1 and Marco Meleti1 1 University Hospital of Parma, Department of Medicine and Surgery, Italy 2 Local Health Authority of Reggio Emilia (IRCCS), Italy Aim. Saliva is a biofluid potentially useful for new approaches based on the quantitative and qualitative analysis of biomarkers. Potential applications of salivary biomarkers include early diagnosis, prognosis, follow-up and management of oral and systemic diseases. Saliva can be easily collected, in a non-invasive way, and stored for the analysis with a minimum handling, when compared to other biofluids. About 70% of whole saliva (WS) is produced by parotid glands (parotid saliva, PS). Submandibular glands are responsible for the production of about 25% of the fluid, while sublingual glands produce the remaining 5% (submandibular/sublingual saliva - SMS). Water accounts for up to 99% of saliva composition. The remaining 1% is composed of a variety of electrolytes, including sodium, potassium, calcium, magnesium, bicarbonate and phosphates. A small amount of nucleic acids, proteins and metabolites are also found. WS, being the product of the mixing of PS, SMS, minor salivary glands saliva and crevicular fluid and being massively contaminated by bacteria, food and environmental molecules may possibly not reflect the composition of “pure” saliva (namely the biofluid, at its release from the salivary ducts, before its diffusion in the oral cavity). Saliva composition may vary in association to several factors such as age, gender, circadian rhythm, diet, medication and environmental exposure. Pathologies also contribute to quantitative / qualitative salivary variation. Most of the chemical, genomic, transcriptomic and proteic features of saliva have been described in detail. Instead, the nature and concentration of salivary metabolites have still poorly been evaluated. Some researches have been conducted, leading to the identification of about nine hundred metabolites. Such studies also correlated the metabolic concentration with different physiological and pathological conditions. Data on salivary metabolomics are available only for WS, the metabolic composition of the other salivary types (PS and SMS) having not yet been described. The characterization of the metabolic profiles and the evaluation of differences between PS, SMS and WS, might help to identify which, among these saliva sub-types shows more similarity to blood. This would hypothetically allow the development of precise salivary diagnostic techniques, potentially suitable as adjunct to hematological sampling and analysis. The aims of the present study are: - to report the differences in the metabolic composition between WS, PS, SMS collected by one healthy subject; - to highlight the differences between the metabolic composition of different saliva sub-types and serum, in order to identify which among WS, PS and SMS, reflects the blood conditions. Materials and Methods. Saliva subtypes examined in the present study was collected by a 24 year-old volunteer healthy woman. No recent pathologies or medications were reported on anamnesis. The subject had a DMFT score indicating the presence of less than 5 teeth with active caries or with at least 1 tooth with decaying caries; she also had a PSR score inferior to 4 in all the sextants; no inflammatory disorders of the oral mucosa or potentially malignant lesions could be detected at clinical inspection. Before saliva collection, the patient rinsed her mouth with water for one minute. Collection of PS. The patient was placed in supine position, with the head slightly rotated, in order to easily access the area of Steno duct. Cotton rollers and two dental suction units were placed both in the floor of the mouth and in the vestibule. A sterile sponge was placed on the Steno duct papilla. The sponge was squeezed every 3 minutes in a Eppendorf tube through a sterile syringe;. 4 mL of PS were collected. Collection of SMS. The patient, seated on the dental chair, was positioned with the head and back perpendicular to the floor. Cotton rollers were placed at the level of the oral vestibule. Saliva was collected in the floor of the mouth by suction, through a needle-free sterile syringe, once every minute. The content of the syringe was poured into a sterile tube. The collection finished when 4 mL of saliva were reached. Collection of WS. The subject was positioned on the dental chair with her back straight, perpendicular to the floor. She did not swallow saliva for 1 minute. Subsequently, the entire amount of fluid present in the mouth was spit into a sterile container. The collection finished when 4 mL of saliva were collected. During saliva collection, tubes were placed on ice (4 °C). Until centrifugation, each sample was stored in a refrigerated portable container. The collection of 5 mL of blood in a test tube without anticoagulant was performed. Saliva samples were immediately centrifuged at 25600 g for 15 min at 4 °C. Following centrifugation, the supernatant was separated from the pellet. Blood sample was processed after one hour of coagulation. The serum was obtained following centrifugation at 3000 g x 15 min at 4 °C. All samples were stored at -80 °C,. The metabolic profiles of the biofluids were determined by proton nuclear magnetic resonance (1H-NMR) in a JEOL 600 MHz ECZ600R spectrometer at the Interdepartmental Measurements Center (CIM) of the University of Parma. The samples were prepared with the addition of phosphate buffer and TSP (trimethylsilylpropane acid) as internal reference. The identification and quantification of the metabolites were performed using the Chenomx NMR suite 7.6 software. and MestreNova. Results. Thirthy-four metabolites were identified in the serum sample, 31, 27, 21 in WS, PS and SMS, respectively. WS resulted to be the richest in metabolites. Most important differences among salivary samples were found among the concentration of the following metabolites: 1) acetate has a higher concentration in WS (20 times higher than serum); 2) urea in PS has a concentration almost doubled than serum and 4 times higher than WS; 3) interestingly, glucose is not present in WS, while it is found in PS and in SMS; 4) quantity of lactate is very high in PS and very low in WS and in SMS; 5) formic acid is not present in WS; 6. acetone is very high in WS, and completely absent in PS and SMS. Discussion. On the basis of the very preliminary results of the present research and, taking into account that, to the best of our knowledge, this is the first study that describes the metabolic differences between WS, PS and SMS, we can only put forward a serier of hyoptheses to explain our findings. Particularly, the absence of glucose in WS and the presence in PS and SMS could be explained taking into account the action of microorganisms present in the oral cavity such as Streptococci, Spirochetes, Lactobacilli, Actinomycetes. The metabolism of these bacteria depends on the presence of sugars (including monosaccharides such as glucose). The presence of acetate in WS and the almost absence of this in PS and in SMS could be due to the phenomenon of bacterial antagonism. In the oral cavity there are bacteria, such as S. sanguis, S. mutans, and S. salivarius which produce acetic acid which is toxic to many other microorganisms. The very high concentration of urea in PS compared to the WS and serum could be explained by the conversion of such molecule into ammonia by the urease-positive bacteria residing in the oral cavity. Our study confirms that salivary metabolic analysis seems to be a promising field for diagnosis of local and systemic diseases. References 1. Malamud D., Rodriguez-Chavez IR. Saliva as a diagnostic fluid. Dent Clin North Am.: 2011; 55: 159–78. 2. Takeda I, Strech C, Barnaby P, et al. Understanding the human salivary metabolome. NMR Biomed. 2009; 22: 577-84. 3. Zerihun T. Dame, Farid Aziat, Rupasri Mandal, Ram Krishnamurthy, Souhaila Bouatra, Shima Borzouie, An Chi Guo, Tanvir Sajed, Lu Deng, Hong Lin, Philip Liu, Edison Dong, David S. Wishart. The human saliva metabolome. Metabolomics:2015; 11 (6), 1864–83. Keywords: Saliva, Salivary Glands, diagnosis, salivary biomarkers, Metabolomics Conference: 5th National and 1st International Symposium of Italian Society of Oral Pathology and Medicine., Ancona, Italy, 19 Oct - 20 Oct, 2018. Presentation Type: Poster Presentation Topic: Salivary Research Citation: Antonelli R, Pertinhez T, Pezzi M, Ghezzi B, Vescovi P and Meleti M (2019). Differences in metabolic profile between submandibular, parotid and whole saliva: a new concept in salivary diagnostics.. Front. Physiol. Conference Abstract: 5th National and 1st International Symposium of Italian Society of Oral Pathology and Medicine.. doi: 10.3389/conf.fphys.2019.27.00034 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 01 Nov 2018; Published Online: 09 Dec 2019. * Correspondence: Dr. Rita Antonelli, University Hospital of Parma, Department of Medicine and Surgery, Parma, Emilia-Romagna, Italy, marcomascitti86@hotmail.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rita Antonelli Thelma Pertinhez Margherita Eleonora Pezzi Benedetta Ghezzi Paolo Vescovi Marco Meleti Google Rita Antonelli Thelma Pertinhez Margherita Eleonora Pezzi Benedetta Ghezzi Paolo Vescovi Marco Meleti Google Scholar Rita Antonelli Thelma Pertinhez Margherita Eleonora Pezzi Benedetta Ghezzi Paolo Vescovi Marco Meleti PubMed Rita Antonelli Thelma Pertinhez Margherita Eleonora Pezzi Benedetta Ghezzi Paolo Vescovi Marco Meleti Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.