Dietary Nitrate and the Oral Microbiome in Children With Primary Hypertension

Primary hypertension (PH) in children and adolescents is increasingly recognized as a significant public health concern. Epidemiological data from multiple cohorts, including the Young Finns Study and other long-term follow-ups, demonstrate that elevated systolic blood pressure beginning in adolescence predicts endothelial dysfunction, increased carotid intima-media thickness, and a higher lifetime risk of cardiovascular morbidity. Children with PH frequently show early subclinical changes such as impaired flow-mediated dilation, increased arterial stiffness, heightened sympathetic activity, and altered nitric oxide (NO) bioavailability. Despite these findings, the early biological mechanisms that contribute to PH in youth remain insufficiently characterized, and non-invasive biomarkers for early detection are limited. Recent advances in pediatric cardiovascular science suggest that the nitrate-nitrite-nitric oxide (NO₃- → NO₂- → NO) enterosalivary pathway may play a previously underappreciated role in blood pressure regulation. This pathway relies heavily on commensal oral bacteria capable of reducing dietary nitrate to nitrite, which is then swallowed and converted to NO systemically. Any disruption in this oral microbial nitrate-reducing capacity, whether due to dysbiosis, impaired oral hygiene, inflammation, dietary differences, or ecological shifts during adolescence may diminish NO bioavailability and subsequently raise blood pressure. Several adult studies have shown that antibacterial mouthwashes, changes in oral microbiota composition, and reduced salivary nitrate-reducing activity lead to measurable increases in systolic and diastolic blood pressure. However, pediatric data remain extremely scarce, with no comprehensive metagenomic investigation of oral microbiota in children with PH published to date. This study is therefore designed to characterise, in detail, the relationships between (i) salivary microbiota composition, (ii) nitrate/nitrite conversion capacity, (iii) salivary biochemical characteristics, and (iv) blood pressure profiles in adolescents aged 12-18 years diagnosed with primary hypertension. Importantly, the project employs non-invasive sampling methods (stimulated and unstimulated saliva), enabling participation from children who may not tolerate venipuncture or invasive sampling procedures, and providing a practical model for future screening tools. 1. Scientific Rationale and Background 1.1. Pediatric Hypertension Primary hypertension in youth was once considered rare but now affects up to 3-5% of adolescents globally. It is strongly associated with obesity, insulin resistance, sympathetic overactivation, endothelial dysfunction, and chronic inflammatory states. Although pharmacological treatment is increasingly used, current pediatric guidelines emphasise early identification and non-pharmacological management strategies. 1.2. Oral Microbiota and Blood Pressure The oral cavity hosts over 700 bacterial species, many of which participate in nitrate metabolism. Key nitrate-reducing genera include Veillonella, Neisseria, Rothia, Actinomyces, and Prevotella. Disturbances in these communities due to diet, oral hygiene, frequent mouthwash use, periodontal inflammation, or underlying systemic disease may reduce the efficiency of nitrate reduction. Several human trials in adults have demonstrated that: mouthwash-mediated suppression of nitrate-reducing bacteria acutely increases blood pressure, dietary nitrate supplementation raises salivary nitrite levels and lowers systolic BP, individuals with lower nitrate-reducing capacity have higher resting BP. However, children fundamentally differ from adults in oral ecology, immune maturation, hormonal shifts during puberty, diet, and salivary gland physiology. Therefore, adult data cannot be extrapolated to pediatric populations. 1.3. Nitric Oxide, Vascular Function, and Pediatric Risk NO is vital for vascular homeostasis. Reduced NO availability contributes to endothelial dysfunction, impaired vasodilation, increased vascular tone, and early arterial stiffness. Several pediatric studies have demonstrated: elevated basal NO metabolites in obese children due to chronic low-grade inflammation, disrupted NO pathways in children with early metabolic abnormalities, correlations between salivary nitrite levels and systemic metabolic markers. Yet, no study has simultaneously examined oral microbiome composition + nitrate reductase activity + pediatric hypertension in a unified framework. 2. Study Objectives and Hypotheses Primary Objective To determine whether adolescents with primary hypertension exhibit alterations in oral microbiota composition and reduced salivary nitrate-reducing capacity compared with normotensive controls. Secondary Objectives To quantify salivary nitrate, nitrite, pH, flow rate, total protein, lysozyme activity and correlate these with blood pressure levels. To explore associations between oral microbial diversity (alpha/beta diversity metrics) and hypertension status. To identify specific bacterial taxa or functional gene pathways (via metagenomic sequencing) associated with impaired nitrate reduction. To evaluate whether salivary microbiome features could serve as non-invasive biomarkers for early vascular risk or masked hypertension. To explore relationships between nitrate-related pathways and anthropometric measures, oral health indices, diet, and lifestyle. Hypotheses Children with PH have distinct oral microbial compositions with lower abundance of nitrate-reducing bacteria. Reduced nitrate-reducing capacity is associated with lower salivary nitrite, altered NO metabolism, and higher systolic BP. Specific microbial signatures may predict masked hypertension and early vascular dysfunction. 3. Study Design This is a observational, cross-sectional, non-interventional clinical study. No therapeutic agents or dietary supplements will be administered. All measurements rely on non-invasive, routine pediatric dental and clinical procedures. 3.1. Participants Two groups will be recruited: Primary Hypertension Group (PH Group) Diagnosed according to AAP and ESH pediatric hypertension criteria. Healthy Control Group Age- and sex-matched. Normotensive based on standardized clinic measurements. 3.2. Sample size Based on prior microbiome variability estimates and effect sizes in adult nitrate-reduction studies, a minimum of 48 participants is planned (24 PH + 24 control). This number is adequate for exploratory metagenomic and metabolite correlations. Power calculations used α=0.05 and β=0.20 for differences in nitrate reduction capacity. 4. Data Collection Procedures 4.1. Blood Pressure Measurement Performed using pediatric cuff-adjusted oscillometric devices validated for clinical use. Measurements taken after 5 minutes of rest, in a seated position, using three repeated readings. 4.2. Clinical Oral Examination Conducted by a trained pediatric dentist. Assessment includes dental caries, gingival status, and plaque presence. 4.3. Saliva Collection (Unstimulated) Unstimulated saliva for baseline nitrate/nitrite measurements, pH, flow rate, total protein. Standardized collection times (morning hours, ≥1 hr after eating, drinking, brushing). 4.4. Laboratory Measurements Biochemical analyses Nitrate and nitrite concentrations via standardized kits (colorimetric methods). Salivary lysozyme activity (innate immunity marker). Total protein via BCA assay. pH via pH strips. Metagenomic DNA Extraction Using a microbiome DNA extraction kit ensuring lysis of both Gram-positive and Gram-negative bacteria. Library Preparation for NGS Metagenomics library preparation kit DNA barcoding kit Library quantification and normalization using Illumina-based quantification kits. Sequencing Performed using Illumina MiSeq 2×300 bp paired-end sequencing. Quality control steps include fragment size profiles, chimera removal, and adapter trimming. Bioinformatics Pipeline FASTQ quality filtering using QIIME2 or similar platforms. Taxonomic classification using SILVA/Greengenes/HOMD reference databases. Functional analysis via PICRUSt2 or equivalent tools. Diversity analyses with Shannon, Simpson, Bray-Curtis metrics. 5. Data Management and Quality Assurance 5.1. Data Validation All collected data will undergo range checks and logical consistency checks (e.g., BP ranges, anthropometric plausibility). Automated scripts will flag inconsistencies for review. 5.2. Source Verification Clinical data will be reviewed against patient charts by trained clinicians. Saliva processing forms will be cross-checked with laboratory data. 5.3. Laboratory QC Use of negative/blank controls during DNA extraction and library preparation. Duplicate samples for \~10% of participants to assess technical variability. Calibration logs for pipettes, thermocyclers, and sequencing equipment. 5.4. Data Security Data stored on institutional encrypted servers behind firewall protection. Only authorized team members will have access. 6. Statistical Analysis Plan 6.1. Primary Analysis Compare microbial diversity and nitrate-reducing capacity between PH and control groups using: Wilcoxon/Mann-Whitney U tests PERMANOVA for beta-diversity Differential abundance analysis (ANCOM, DESeq2) 6.2. Secondary Analyses Spearman correlations between salivary biochemical markers and systolic/diastolic BP. Multiple linear regression adjusting for BMI, diet, oral hygiene habits. Exploratory machine-learning models (random forest) to identify predictive microbial features. 6.3. Missing Data Plan Missing biochemical values handled via multiple imputation or complete case analysis. Missing microbiome data due to low DNA yield will be documented, not imputed. 7. Ethical Considerations This study involves minimal risk. No blood samples, medications, or interventions will be given. Saliva collection is non-invasive and well tolerated. Consent and assent procedures will be conducted according to institutional and national regulations. Ethical approval was obtained from the Istanbul University Faculty of Dentistry Clinical Research Ethics Committee at its meeting dated 14/10/2025 and numbered 206. 8. Potential Impact The project may reveal novel, non-invasive microbial signatures of early vascular risk in adolescents. Findings could support: early screening tools for masked or mild hypertension, dietary nitrate-based preventive strategies, better understanding of the oral microbiome-vascular axis in youth.