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Randomized Controlled Trial
. 2018 Aug 20:124:21-30.
doi: 10.1016/j.freeradbiomed.2018.05.078. Epub 2018 May 25.

Nitrate-responsive oral microbiome modulates nitric oxide homeostasis and blood pressure in humans

Anni Vanhatalo  1 , Jamie R Blackwell  2 , Joanna E L'Heureux  2 , David W Williams  3 , Ann Smith  4 , Mark van der Giezen  2 , Paul G Winyard  5 , James Kelly  2 , Andrew M Jones  2
Affiliations
Randomized Controlled Trial

Nitrate-responsive oral microbiome modulates nitric oxide homeostasis and blood pressure in humans

Anni Vanhatalo et al. Free Radic Biol Med. .

Abstract

Imbalances in the oral microbial community have been associated with reduced cardiovascular and metabolic health. A possible mechanism linking the oral microbiota to health is the nitrate (NO3-)-nitrite (NO2-)-nitric oxide (NO) pathway, which relies on oral bacteria to reduce NO3- to NO2-. NO (generated from both NO2- and L-arginine) regulates vascular endothelial function and therefore blood pressure (BP). By sequencing bacterial 16S rRNA genes we examined the relationships between the oral microbiome and physiological indices of NO bioavailability and possible changes in these variables following 10 days of NO3- (12 mmol/d) and placebo supplementation in young (18-22 yrs) and old (70-79 yrs) normotensive humans (n = 18). NO3- supplementation altered the salivary microbiome compared to placebo by increasing the relative abundance of Proteobacteria (+225%) and decreasing the relative abundance of Bacteroidetes (-46%; P < 0.05). After NO3-supplementation the relative abundances of Rothia (+127%) and Neisseria (+351%) were greater, and Prevotella (-60%) and Veillonella (-65%) were lower than in the placebo condition (all P < 0.05). NO3- supplementation increased plasma concentration of NO2- and reduced systemic blood pressure in old (70-79 yrs), but not young (18-22 yrs), participants. High abundances of Rothia and Neisseria and low abundances of Prevotella and Veillonella were correlated with greater increases in plasma [NO2-] in response to NO3- supplementation. The current findings indicate that the oral microbiome is malleable to change with increased dietary intake of inorganic NO3-, and that diet-induced changes in the oral microbial community are related to indices of NO homeostasis and vascular health in vivo.

Keywords: 16S rRNA sequencing; Ageing; Cardiovascular health; Host-microbe symbiosis; Nitrite; Oral nitrate reduction; Prebiotic.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Participants underwent 10-day supplementation periods with nitrate (~ 12.4 mmol/d) and placebo in a balanced cross-over design. Screening, protocol familiarisation and Salivary Flow Rate Questionnaires (SFR-Q) were completed at baseline. Measurements of plasma nitrite (NO2-) and nitrate (NO3-) concentrations, blood pressure (BP) of the brachial artery, arterial stiffness as carotid-femoral pulse wave velocity (PWV), and the collection of saliva samples for microbiome analysis were undertaken on days 8, 9 and 10 of each supplementation period.
Fig. 2
Fig. 2
Plasma [NO3-] (panel A) and [NO2-] (panel B) were significantly greater after nitrate supplementation (white bars) compared to placebo (black bars) (n = 18). The change (Δ) in plasma [NO3-] between nitrate and placebo conditions was similar in young (n = 9) and old participants (n = 9) (panel C), but Δ[NO2-] was significantly greater in the old compared to young participants (panel D). Error bars indicate standard deviations and black squares (panels C and D) indicate means for young and old participants. *P < 0.05.
Fig. 3
Fig. 3
Mean arterial pressure (MAP; panel A), systolic blood pressure (SBP; panel D), diastolic blood pressure (DBP; panel G) and pulse wave velocity (PWV; panel J) were not different between placebo and nitrate conditions across all participants (n = 18). The old participants (n = 9) showed greater reductions (Δ) in MAP, SBP and DBP between placebo and nitrate conditions than the young participants (n = 9; panels B, E and H), as well as a greater increase in PWV (young n = 9, old n = 8; panel K). ΔMAP, ΔSBP and ΔDBP inversely correlated with the change in plasma [NO2-] relative to nitrate dose (Δ[NO2-]/NO3- dose) (panels C, F and I) and ΔPWV positively correlated with Δ[NO2-]/NO3- dose (panel L). *P < 0.05.
Fig. 4
Fig. 4
The proportions of five main phyla of oral bacteria identified in the saliva samples following 10 days of placebo (PL) and NO3- supplementation (BR). *Difference between PL and BR (P < 0.05).
Fig. 5
Fig. 5
Overall salivary microbiome composition illustrated by non-metric multidimensional scaling (NMDS) analysis. The salivary microbiome composition was different between nitrate (BR) and placebo (PL) conditions (P < 0.05; panel A) but not between young and old participants (P > 0.05; panel B).
Fig. 6
Fig. 6
The Shannon diversity index indicated no statistically significant difference in species diversity between nitrate (BR) and placebo (PL) conditions.
Fig. 7
Fig. 7
The genera (panels A and B) and species (panels C and D) that comprised > 0.01% of all bacteria and showed significant differences (P < 0.05) between nitrate (BR) and placebo (PL) conditions.
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