Intake of Vegetables Rich in Nitrate and Its Effect on the Oral Microbiota of Patients With Periodontitis

Intake of Vegetables Rich in Nitrate and Its Effect on the Oral Microbiota of Patients With Periodontitis

Effect of the Intake of Vegetables Rich in Nitrate on the Nitrate-reducing Capacity and Composition of the Oral Microbiota in Patients With Periodontitis

Vegetables, mainly green leafy ones and some others like beets, are a rich source of nitrate. Nitrate metabolism has shown benefits to the body and there are recent studies that agree that the consumption of nitrates from vegetables can modify the oral microbiota by increasing species with nitrate-reducing capacity, which are also bacteria that are associated with oral health.

Background. Periodontitis (PD) is presented by the imbalance that results in the proliferation of certain pathogenic bacteria that alter the symbiotic community and cause oral dysbiosis. Non-surgical periodontal treatment helps to eliminate the pathogenic plaque present, and it is of interest to seek adjuvant treatments that contribute to an improvement in oral health. The oral microbiota plays an essential role in the human nitro-reduction process, since specific bacteria reduce exogenous nitrate to nitrite, to later convert into nitric oxide within the body. Vegetables, mainly green leafy ones and some others like beets, are a rich source of nitrate. Nitrate metabolism has shown benefits to the body and there are recent studies that agree that the consumption of nitrates from vegetables can modify the oral microbiota by increasing species with nitrate-reducing capacity, which are also bacteria that are associated with oral health. Therefore, our hypothesis maintains that the consumption of vegetables can be an adjunct to the recovery and improvement of the condition in patients receiving non-surgical periodontal treatment. Objective. To evaluate the effect of the consumption of vegetables rich in nitrate on the nitrate-reducing capacity and composition of the oral microbiota in patients with PD. Material and methods. A parallel randomized clinical trial (RCT) will be conducted in 60 PD patients who are candidates for non-surgical periodontal treatment (NSPT) and they will be randomized into two groups. The control group will receive the NSPT and instructions to continue with their usual diet, while the intervention group, in addition to the NSPT, will receive a nutritional intervention to promote the daily consumption of foods rich in nitrate. The nutritional intervention will be based on the theory of the wheel of behavior change to achieve the consumption of vegetables rich in nitrate that provide ~ 350 mg of this compound. Both groups will be evaluated before and after the study: clinical, dental, and nutritional parameters. Likewise, samples will be taken of blood to perform blood chemistry, saliva to perform a nitrate reduction test and quantify the number of nitrates and nitrites, as well as subgingival plaque and tongue scraping, which will be sequenced to obtain the diversity and abundance of nitro-reducing bacteria at the beginning and end of the study in both groups.

This group will receive non-surgical periodontal treatment, however they will not receive any modification to their diet

This group will receive non-surgical periodontal treatment, and also as a treatment of interest, will receive the indication to consume ~ 350 mg of nitrate from vegetables, as well as an accompaniment to achieve this objective.

Nutritional intervention for 10 weeks to increase the consumption of vegetables rich in nitrate. At the beginning of the NSPT on visit 1 (week 2), the first of 5 nutritional interventions will begin with the objective of consuming ~ 350 mg of NO3 daily from vegetables

The composition of the oral microbiota will be before and after the intervention will be evaluated in both study groups using 16S rRNA gene Illumina sequencing. For their analysis, alpha diversity will be evaluated: richness using the Chao1 index and diversity using the Shannon index; beta diversity with PCoA (principal coordinates analysis). In addition, taxonomic assignment will be performed with the DADA2 protocol at both genus and species (ASVs) level, against the SILVA database.

The nitrate-reducing capacity in the mouth will be evaluated before and after the intervention in both study groups. Nitrite and nitrate levels will be quantified in saliva and in a nitrate reduction test. Test strips will be used and measured in a Merck reflectometer, which will provide the mg/L of each metabolite.

Three 24-hour diet recall will be performed and the average nitrate intake in g/day will be evaluated. Tables reporting the average amount of nitrate in food will be used and multiplied by the reported amount of food consumed by the participant.

Periodontal chart is a tool used by dental professionals to evaluate different parameters such as probing depth, clinical attachment level (CAL), bleeding on probing (BP), plaque or calculus (PoC), which help to identify the condition of the periodontal tissues and determine the status of the disease. The measurement of these parameters may give an indication of the extent and severity of the periodontal disease, a Hu Friedy periodontal probe is used for the evaluation, each tooth is divided into six points for study in the vestibular or lingual and palatal aspect, mesial, central and distal. A periodontist will perform the periodontal chart before and after the intervention in both study groups. The online periodontogram of the Spanish Society of Periodontology and Osseointegration (SEPA) will be used: http://sepa.es/periodontograma/index.html

The periodontal probe is inserted along the root surface of the tooth in order to measure the distance from gingival margin to pocket bottom of the periodontal pocket. The values are measured in mm, six sites per tooth, measurements are expressed as averages.

The clinical attachment level provides an estimation of the true periodontal support and is used for monitoring changes in periodontal support over the time. The "Attachment Level" for each site will be calculated based on the following formula: probing depth (mm) less gingival margin (mm) if the cemento-enamel junction (CEJ) is cover to the gingival margin. In situation where the gingival margin is apical to the CEJ, probing depth (mm) sum the distance to CEJ at gingival margin (mm). The values are measured in mm, six sites per tooth for each teeth present in the mouth, measurements are expressed as averages.

The periodontal probe is carefully introduced to the bottom of the pocket and gently moved laterally around the tooth, six points are recorded for each tooth as bleeding present or absent, the measurements are expressed as percentage of bleeding on probing of teeth present in the mouth. The bleeding in each tooth is identified and the values obtained are used to average the bleeding.

The presence of plaque and calculus is evaluated and marked as present or absent on the periodontal chart based on 6 points on the tooth, the measurements are expressed as percentage of plaque or calculus (PoC).

Inclusion Criteria: Men and/or women from 30 to 65 years old Voluntary participation and signing the written informed consent Not receiving nutritional treatment at the time of uptake Availability to attend all scheduled visits Have an indication to receive nonsurgical periodontal treatment (NST) Interdental clinical insertion loss ≥3mm and ≥5mm in the buccal or oral area of two or more non-adjacent teeth Detectable depths ≥ 5mm in two or more teeth Radiographic evidence of periodontal bone loss Exclusion Criteria: Trauma-Induced Gingival Recessions Cervical caries History of bleeding diathesis Pregnant or nursing women BMI <18.5 kg / m2 History of any of the following self-reported diseases: type 2 diabetes mellitus, high blood pressure, kidney or liver disease, inflammatory bowel diseases, autoimmune diseases and / or use of medications to control these diseases. Having or presenting a high risk of infection by tuberculosis, hepatitis B or Human Immunodeficiency Virus (HIV) Use of antibiotics (including antibiotic oral rinse) 6 months prior to or during the study Having received specialized periodontal treatment in the previous 6 months. Smoking habit Require an interpreter or not understand Spanish to attend or provide written material. Patients diagnosed with COVID-19.