Dietary nitrate improves vascular function in patients with hypercholesterolemia: a randomized, double-blind, placebo-controlled study

Abstract

Background: The beneficial cardiovascular effects of vegetables may be underpinned by their high inorganic nitrate content.

Objective: We sought to examine the effects of a 6-wk once-daily intake of dietary nitrate (nitrate-rich beetroot juice) compared with placebo intake (nitrate-depleted beetroot juice) on vascular and platelet function in untreated hypercholesterolemics.

Design: A total of 69 subjects were recruited in this randomized, double-blind, placebo-controlled parallel study. The primary endpoint was the change in vascular function determined with the use of ultrasound flow-mediated dilatation (FMD).

Results: Baseline characteristics were similar between the groups, with primary outcome data available for 67 patients. Dietary nitrate resulted in an absolute increase in the FMD response of 1.1% (an ∼24% improvement from baseline) with a worsening of 0.3% in the placebo group (P < 0.001). A small improvement in the aortic pulse wave velocity (i.e., a decrease of 0.22 m/s; 95% CI: -0.4, -0.3 m/s) was evident in the nitrate group, showing a trend (P = 0.06) to improvement in comparison with the placebo group. Dietary nitrate also caused a small but significant reduction (7.6%) in platelet-monocyte aggregates compared with an increase of 10.1% in the placebo group (P = 0.004), with statistically significant reductions in stimulated (ex vivo) P-selectin expression compared with the placebo group (P < 0.05) but no significant changes in unstimulated expression. No adverse effects of dietary nitrate were detected. The composition of the salivary microbiome was altered after the nitrate treatment but not after the placebo treatment (P < 0.01). The proportions of 78 bacterial taxa were different after the nitrate treatment; of those taxa present, 2 taxa were responsible for >1% of this change, with the proportions of Rothia mucilaginosa trending to increase and Neisseria flavescens (P < 0.01) increased after nitrate treatment relative to after placebo treatment.

Conclusions: Sustained dietary nitrate ingestion improves vascular function in hypercholesterolemic patients. These changes are associated with alterations in the oral microbiome and, in particular, nitrate-reducing genera. Our findings provide additional support for the assessment of the potential of dietary nitrate as a preventative strategy against atherogenesis in larger cohorts. This trial was registered at clinicaltrials.gov as NCT01493752.

Keywords: endothelium; microbiome; nitric oxide; vascular; vegetable.

FIGURE 1

CONSORT flowchart of study. CONSORT, Consolidated Standards of Reporting Trials.

FIGURE 2

Dietary nitrate elevates plasma, salivary, and urinary nitrite and nitrate concentrations in hypercholesterolemic patients. Mean ± SD effects of 6 wk of dietary nitrate consumption (250 mL nitrate-rich juice/d) or placebo consumption (250 mL nitrate-depleted juice/d) on nitrite and nitrate concentrations in plasma (A and B), saliva (C and D) and urine (E and F), respectively. n = 33 in the nitrate group; n = 32 in the placebo group. ****Significant, P < 0.001 (1-factor ANOVA with Bonferroni posttests). There were no significant differences in any comparisons between baseline measures in the nitrate and placebo groups.

FIGURE 3

Dietary nitrate improves vascular function in hypercholesterolemic patients. Mean ± SD effects of 6 wk of dietary nitrate consumption (250 mL nitrate-rich juice/d) or placebo consumption (250 mL nitrate-depleted juice/d) on FMD. n = 33 in the nitrate group; n = 32 in the placebo group. Baseline and 6-wk data are shown for groups before and after intake of nitrate-rich juice and nitrate-depleted placebo juice. P values shown are for within-group comparisons with the use of paired t test for the comparison of the baseline FMD with the response after 6 wk of intervention. Comparison between groups are shown with P values for the change from baseline with the use of an unpaired t test. FMD, flow-mediated dilatation.

FIGURE 4

Associations between plasma nitrite concentrations and vascular function measures. Changes were determined from baseline to the 6-wk time point in FMD relative to changes in plasma nitrite concentration (A) and changes in blood pressure (SBP) (B). (C) Changes in vascular stiffness aortic PWV are plotted against changes in SBP. Associations were determined with the use of a Pearson’s correlation coefficient assessment. The data show values for n = 33 in the nitrate group and n = 32 in the placebo group for each correlation analysis. FMD, flow-mediated dilatation; SBP, systolic blood pressure. PWV, pulse wave velocity.

FIGURE 5

Dietary nitrate decreases platelet monocyte aggregate concentrations. Mean ± SD effects of 6 wk of dietary nitrate consumption (250 mL nitrate-rich juice/d) or placebo consumption (250 mL nitrate-depleted juice/d) on flow-cytometry measures of PMA (A) and the percentage of PMA formation for groups before and after intake of nitrate-rich juice and placebo juice (B). (C) Change in % PMA formation over 6 wk in the 2 groups expressed as mean (95% CI). Data shown are n = 25 for the nitrate group and n = 27 for the placebo group. **Significant for within-group comparisons of baseline compared with postnitrate consumption, P < 0.01 (paired Student’s t test). *Significant for the comparison between groups, P < 0.05 (unpaired t test). PMA, platelet-monocyte aggregate.

FIGURE 6

Dietary nitrate results in changes in the oral microbiome. The use of a bacterial community profile analysis by means of the mothur pipeline (16S ribosomal RNA gene) identified 78 different OTUs, the proportions of which were altered by dietary nitrate treatment. The statistical analysis was conducted with the use of an AMOVA (38) for assessment of the change between groups in the oral microbial community (39). The AMOVA gave P < 0.001 for the within-group comparison between baseline and post-nitrate and P = 0.001 for the between-group comparison of post-placebo compared with post-nitrate. Of these OTUs, those that had their numbers increase post-treatment and that made up >1% of the post-treatment community were Neisseria flavescens and Rothia mucilaginosa. (A) Plot depicts a principal coordinate analysis that was based on the ThetaYC metric, which compared the structure of the communities (PC1 = 34.6% of variance explained; PC2 = 56.2%). Colored circles represent the 2 groups of the study. Blue and green circles represent baseline and after 6 wk of placebo intake, respectively, and purple and red circles represent baseline and after 6 wk of once-daily intake (5 mmol) of dietary nitrate juice, respectively. (B) Representation of the relative abundances of R. mucilaginosa and N. flavescens at baseline and postnitrate or postplacebo treatment of 6 wk. Data are shown for baseline and 6-wk values for n = 16 in the nitrate group and n = 14 in the placebo group. Solid lines denote group means, and dotted lines denote group medians. AMOVA, analysis of molecular variance; OTU, operational taxonomic unit.