The blood pressure lowering effect of beetroot juice is impaired in periodontitis and recovered after periodontal treatment

Abstract

We have previously demonstrated that subgingival levels of nitrate-reducing bacteria, as well as the in vitro salivary nitrate reduction capacity (NRC), were diminished in periodontitis patients, increasing after periodontal treatment. However, it remains unclear if an impaired NRC in periodontitis can affect systemic health. To determine this, the effect of nitrate-rich beetroot juice (BRJ) on blood pressure was determined in 15 periodontitis patients before and 70 days after periodontal treatment (i.e., professional mechanical plaque removal, oral hygiene instruction, and subgingival instrumentation), as well as in a healthy control group of 15 individuals. Additionally, subgingival and tongue samples were taken to analyse the bacterial composition with Illumina sequencing of the 16S rRNA gene. In healthy individuals, the systolic and diastolic blood pressure (SBP and DPB) decreased significantly (both P < 0.01) 90 min after BRJ intake, but not in periodontitis patients. However, after periodontal treatment, this blood pressure-lowering effect was recovered (P < 0.05 for SBP; P < 0.01 for DBP). Lower levels of salivary nitrate after identical doses of BRJ intake indicated a potentially higher NRC in healthy individuals (P < 0.05). Periodontitis-associated bacteria decreased in tongue and subgingival samples after periodontal treatment (P < 0.01). In contrast, nitrate-reducing bacteria were associated with health in both habitats, but increased only in subgingival plaque after periodontal treatment (P < 0.001). This is the first study showing that periodontitis could limit the blood-pressure lowering effects of nitrate reduction by the oral microbiota. We propose that an impaired NRC represents a potential link between periodontitis and systemic conditions, which should be confirmed in future randomized controlled trials. Future work should also aim to determine if nitrate prebiotic supplementation and/or tongue cleaning could improve the treatment of periodontitis and its associated comorbidities.

Fig. 1. Blood pressure pre- and post-beetroot juice.

The effect of beetroot juice on the systolic blood pressure (a), diastolic blood pressure (b), and heart rate (c) was determined in 15 healthy individuals and 15 periodontitis patients before (BL) and 70-days after (D70) periodontal treatment. The light blue bars are the parameters before beetroot juice (pre-BRJ) intake and the dark blue bars 1.5 hours after beetroot juice intake (1.5 h post-BRJ). SBP: systolic blood pressure. DBP: diastolic blood pressure. HR: heart rate. BRJ: beetroot juice. *p < 0.05 and **p < 0.01 according to a paired or unpaired t-test for parametric comparisons.

Fig. 2. Comparison of measurements before and 1.5 h after BRJ consumption.

The effect of beetroot juice on salivary nitrate (a) and nitrite (b), salivary flow (c) and pH (d) was determined in 15 healthy individuals and 15 periodontitis patients before (BL) and 70-days after (D70) periodontal treatment. The light blue bars are the parameters before beetroot juice (pre-BRJ) intake and the dark blue bars 1.5 h after beetroot juice intake (1.5 h post-BRJ). NO₃^-: nitrate. NO₂^-: nitrite. μl: microliters. Min: minute. Mg: milligrams. L: liter. Statistics: *p < 0.05 and **p < 0.01 according to an unpaired t-test or paired t-test for parametric comparisons and a Wilcoxon test for nonparametric comparisons.

Fig. 3. Alpha diversity of subgingival plaque and tongue samples at the species level.

Comparing Shannon, ACE and Chao1 indexes in healthy individuals (green bars) and periodontitis patients at baseline (BL, light blue bars) and post-treatment (D70, dark blue bars). The alpha diversity of subgingival plaque (a–c) and tongue (d–f) samples is plotted. Statistics according to an unpaired or paired t-test, *p < 0.05 and **p < 0.01, ***p < 0.001.

Fig. 4. Beta diversity of subgingival plaque and tongue samples at the species level.

Canonical Correspondence Analysis (CCA) comparing the bacterial composition of healthy individuals (blue) and periodontitis patients at baseline (BL, red) and 70 days after treatment (D70, green) at the species taxonomic level, as revealed by 16S rRNA gene Illumina sequencing. The CCA plots of subgingival plaque (a) and tongue (b) samples are shown. Tables below show comparisons between groups and CCA and Adonis p values.

Fig. 5. Relative bacterial abundances in subgingival plaque and tongue coating.

Bar graphs show the top 30 most abundant species in healthy individuals (H) and periodontitis patients before (BL) and after periodontal treatment (D70). Relative abundances are shown in subgingival (a) and tongue (b) samples.

Fig. 6. Log₂ fold changes of all significantly different species in subgingival plaque and tongue samples.

Bar graphs show comparisons between healthy individuals (H) and periodontitis patients at baseline (BL) and 70 days after treatment (D70). The groups that are compared are H and BL (a), and D70 and BL (b) in subgingival plaque, as well as H and BL (c), D70 and BL (d), and h and D70 (e) in tongue coating. Blue bars (positive values) indicate species which increased significantly after treatment or are more abundant in health; red bars (negative values) indicate species which significantly decreased after treatment or are more abundant in disease. To calculate the ratios, relative abundances were used. All plotted organisms were significantly different (adjusted p-values < 0.05) between two groups after standardization of the compositional data by ANCOM-BC. Organisms marked with NA indicate that they could not be assigned at the species level.

Fig. 7. Changes in nitrate-reducing bacteria and periodontitis-associated species.

Bar graphs show the transformed abundance of bacterial groups in subgingival plaque (a–d) and tongue samples (e–h), as estimated by high-throughput sequencing of the 16 S rRNA gene. The relative abundances of species were transformed with ANCOM-BC and grouped in nitrite-producers (a, e), confirmed nitrate reducers (b, f), periodontitis-associated species (c, g) and red complex (d, h) as done previously. *p < 0.05, **p < 0.01, ***p < 0.001 according to a Wilcoxon test (unpaired comparisons) o Mann-Whitney-U test (paired comparison).