Intake of Nitrate-rich Vegetables and Their Effect on Inflammatory Molecules and Oral Microbiota of Patients With Periodontitis

Effect of Nitrate-rich Vegetable Intake on Inflammatory Molecule Levels, Nitrate-reducing Capacity and Oral Microbiota Composition 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.

Study Overview

• Status: Recruiting
• Conditions: Periodontitis; Periodontal Diseases
• Intervention / Treatment: Dietary supplement: Intake of nitrate rich vegetables

Detailed Description

Background. Periodontitis (PD) is a chronic, multifactorial and inflammatory disease caused by the imbalance resulting from the proliferation of certain pathogenic bacteria that alter the symbiotic community and cause oral dysbiosis. Non-surgical periodontal treatment (NSPT) helps to eliminate the pathogenic plaque present and it is of interest to look for adjuvant treatments that contribute to an improvement in oral health, given that, with the passage of time, a recolonization of pathogenic bacteria has been observed, as well as a relapse in patients. The oral microbiota plays a key role in the nitrate reduction process in humans, as certain bacteria reduce exogenous nitrate to nitrite, which is subsequently converted in the body to nitric oxide. Vegetables, mainly green leafy vegetables and some others such as beets, are a rich source of nitrate. Nitrate metabolism has shown benefits for the body and recent studies agree that consumption of nitrates from vegetables can positively modify the oral microbiota. Therefore, we hypothesize that the consumption of vegetables may be an adjuvant in the recovery and improve the condition of patients receiving NSPT and possibly prevent relapse.

Objective. To evaluate the effect of nitrate-rich vegetable intake on salivary levels of inflammatory molecules, nitratoreductive capacity and oral microbiota composition in patients with PD.

Material and methods. A parallel randomized clinical trial (RCT) will be conducted in 60 PD patients who are candidates for 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 vegetables 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. Blood samples will also be taken for blood chemistry and saliva to perform a nitratoreduction test, quantify the amount of nitrates and nitrites and determine the levels of inflammatory molecules, as well as subgingival plaque and tongue scraping, from which DNA will be extracted to be sequenced and to obtain the diversity and abundance of bacteria at the beginning, end of the RCT, 6 months and 12 months later in both groups.

• Study Type: Interventional
• Enrollment (Estimated): 60
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Lucrecia Carrera, PhD; Phone Number: 34240 +523310585200; Email: lucrecia.carrera@academicos.udg.mx
• Study Contact Backup: Name: Nydia S Sanchez, Bachelor; Phone Number: +523311777088; Email: nydia.sanchez0207@alumnos.udg.mx

Study Locations

• Mexico
  • Jalisco
    • Guadalajara, Jalisco, Mexico, 44340
      • Recruiting
      • Integral Dental Clinics CUCS UDG
      • Contact: Alondra Ruiz, PhD; Phone Number: +523312467525; Email: alondracrg80@gmail.com

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 30 years to 65 years (Adult, Older Adult)
• Accepts Healthy Volunteers: No

Description

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 (NSPT)
• 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.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Treatment
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Single

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
No Intervention: Control; This group will receive non-surgical periodontal treatment, however they will not receive any modification to their diet
Experimental: Intervention; 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.	Dietary supplement: Intake of nitrate rich vegetables; 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 nitrate daily from vegetables

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Composition of the oral microbiota	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.	10 weeks, 6 and 12 months
Evaluation of nitrate-reducing capacity	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.	10 weeks, 6 and 12 months
Dietary intake of nitrate	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.	10 weeks, 6 and 12 months
Periodontal chart evaluation	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	10 weeks, 6 and 12 months
Determination of salivary inflammatory molecules	To obtain the saliva sample, the patient will be provided with a sterile 15 ml tube to deposit the accumulated saliva for 5 minutes, this time will be to obtain the largest possible amount of saliva. The saliva sample will not be stimulated (drooling or salivating). Once 3 ml are obtained, the donation will be finished. The saliva will be handled in a sterile area, 40 µl of metalloproteinase inhibitor will be added for each milliliter of saliva to avoid degradation of the molecules of interest, then it will be centrifuged at 4°C at 3,500 rpm for 10 minutes and, once the centrifugation is finished, 250 µl will be aliquoted in 1.5 ml microtubes (a total of 4 tubes) for the determination of the molecules with ELISA kit.	10 weeks, 6 and 12 months

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Probing depth (PD)	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.	10 weeks
Clinical attachment level (CAL)	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.	10 weeks
Bleeding on probing (BP)	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.	10 weeks
Plaque or calculus (PoC)	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).	10 weeks

Collaborators and Investigators

• Sponsor: University of Guadalajara
• Investigators: Principal Investigator: Lucrecia Carrera, PhD, University of Guadalajara

Study record dates

Study Major Dates

• Study Start (Actual): June 23, 2021
• Primary Completion (Estimated): December 31, 2025
• Study Completion (Estimated): December 31, 2025

Study Registration Dates

• First Submitted: November 26, 2021
• First Submitted That Met QC Criteria: February 8, 2022
• First Posted (Actual): February 9, 2022

Study Record Updates

• Last Update Posted (Actual): August 16, 2024
• Last Update Submitted That Met QC Criteria: August 13, 2024
• Last Verified: August 1, 2024

More Information

Terms related to this study

• Keywords: Nitrates; Periodontitis; Vegetables; Oral Microbiota; Inflammatory molecules
• Additional Relevant MeSH Terms: Stomatognathic Diseases; Mouth Diseases; Periodontitis; Periodontal Diseases
• Other Study ID Numbers: CI-02721

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No