The dose–response effects of nitrate-rich beetroot ingestion on cardiovascular and endothelial function: a randomised controlled trial

Abstract

Dietary nitrate (NO₃⁻) supplementation has been reported to improve cardiovascular health, but beyond its effects on brachial artery blood pressure (BP), dose–response effects on other cardiovascular variables are unclear. This study assessed the effects of three acute NO₃⁻ doses (200 mg, 400 mg, 800 mg NO₃^--rich beetroot powder) on brachial and aortic BP, arterial stiffness and macrovascular endothelial function, in a double-blind, randomised, crossover design. Cardiovascular variables and venous blood samples were measured prior to (control) and 2.5 h post supplement ingestion. Dietary NO₃⁻ supplementation increased plasma [NO₃⁻] and plasma [nitrite] but had no effect on cyclic guanosine monophosphate (cGMP) concentration. Arterial stiffness markers improved following all NO₃⁻ doses, with no between-dose differences. However, endothelial function only improved following 400 mg (+3.07% compared to control) and aortic systolic BP only improved following 800 mg (−4 mmHg compared to control) dietary NO₃⁻ supplementation. Acute NO₃⁻ ingestion improved some cardiovascular risk factors, including arterial stiffness, macrovascular endothelial function and aortic systolic BP with different dose–response effects, but had no effect on brachial BP or plasma [cGMP]. These findings improve our understanding of NO₃⁻ supplementation and cardiovascular function in healthy adults.

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Introduction

Cardiovascular disease (CVD) is the leading cause of death worldwide, with endothelial dysfunction, arterial stiffness and hypertension established risk factors for CVD morbidity and mortality.^1,2 Nitric oxide (NO) is a multifaceted physiological signalling molecule in the cardiovascular system, with reduced capacity for NO synthesis implicated in the aetiology of endothelial dysfunction, arterial stiffness and hypertension, and therefore, CVD.^3,4 Historically, NO production was attributed, exclusively, to NO synthase (NOS) enzymes, with nitrate (NO₃⁻) and nitrite (NO₂⁻) considered as biologically inert derivatives of NO oxidation.⁵ It has since been demonstrated that NO₃⁻ and NO₂⁻ can be recycled back to NO via the NO₃⁻–NO₂⁻–NO pathway.⁶ Orally ingested dietary NO₃⁻ is absorbed almost entirely through enterocytes in the small intestine where it enters systemic circulation.⁷ However, the majority is then extracted by the kidneys and excreted in urine (∼60%) in first-pass metabolism. Nonetheless, ∼25% of ingested NO₃⁻ is taken up into the salivary glands and secreted into the oral cavity where it is reduced to NO₂⁻ via NO₃⁻-reducing bacteria. In the stomach, NO₂⁻ is then reduced to NO and other reactive nitrogen intermediates. As a result, there has been great interest in dietary supplementation with inorganic NO₃⁻, which is abundant in beetroot (BR) and green leafy vegetables, as a nutritional NO precursor and cardioprotective nutrient.^8,9 It has been reported that NO₃⁻ supplementation can lower blood pressure (BP) and arterial stiffness and improve endothelial function in healthy adults² and in people with chronic disease,¹⁰ offering broad support for the cardioprotective potential of this nutritional intervention.¹¹

Brachial artery BP has been the most frequently evaluated indicator of cardiovascular function following NO₃⁻ supplementation, though the reported effects have been inconsistent. Indeed, whilst some previous studies have demonstrated 3–9 mmHg reductions in systolic BP (SBP) and 3–6 mmHg reductions in diastolic BP (DBP) following acute ingestion of ∼330–1488 mg NO₃⁻ in healthy individuals,^12–17 other studies have shown no change in SBP or DBP following acute ingestion of ∼400–830 mg NO₃⁻ in this population group.^18–20 From a dose–response perspective, Wylie et al.¹⁴ observed that SBP was reduced after acute ingestion of ∼260 mg NO₃⁻, decreased to a greater extent with ∼520 mg NO₃⁻, but showed no further reduction with a higher dose of ∼1040 mg NO₃⁻, indicating a plateau in the SBP-lowering response.

Compared to brachial BP, the effects of NO₃⁻ ingestion on aortic BP, arterial stiffness variables, and macrovascular endothelial function are less clear. In healthy participants and in individuals with chronic conditions, aortic SBP has been reduced by up to 6 mmHg following a single dose of ∼500–820 mg NO₃⁻ (ref. 20–22), but another study reported no effect.²³ Similarly, it has been reported that arterial stiffness does not change^20,24 or is reduced (improved) by up to 3.3%^2,25 following acute ingestion of ∼830–1533 mg NO₃⁻. Additionally, and despite being an established CVD risk factor,¹ relatively few studies have investigated the effect of NO₃⁻-rich BR supplementation on macrovascular endothelial function.^10,26 Flow mediated dilation (FMD) is an important marker of NO-mediated endothelial function, which has been reported to increase by ∼1–3% following acute ingestion of ∼310–1400 mg NO₃⁻ in some studies,^17,27–30 however an acute dose of ∼512 mg potassium NO₃⁻ had no effect on FMD in healthy young adults in another study.³¹ Interpreting the results of studies investigating the effect of NO₃⁻ on cardiovascular function is complicated by inter-study differences in variables which can alter the efficacy of NO₃⁻ supplementation to improve cardiovascular health variables (BP, arterial stiffness and endothelial function), including NO₃⁻ dose^12,14,24 and form of supplement,^16,32,33 and the population.^34–36 Previous studies assessing effects on brachial BP have suggested that ingestion of higher NO₃⁻ doses does not necessarily translate to greater improvements in cardiovascular function.^11,14 Nonetheless, a recent meta-analyses including subgroup comparisons reported an improvement in arterial stiffness exclusively following NO₃⁻ doses >400 mg.² However, the dose–response effect of acute NO₃⁻ ingestion on macrovascular endothelial function, aortic BP and arterial stiffness has yet to be investigated. This is of importance as the prognostic value of aortic BP, arterial stiffness, and macrovascular endothelial function is increasingly recognised,^1,37 making it relevant to establish effective NO₃⁻ doses to improve these variables.

Although acute NO₃⁻ ingestion appears to improve cardiovascular health markers,^2,38 the mechanisms that underlie such effects are not entirely clear. It is generally postulated that beneficial effects of NO₃⁻ ingestion on the cardiovascular system are NO mediated.³ Classically, NO signalling in the vascular system was considered to function via the NO-cyclic guanosine monophosphate (cGMP)-protein kinase G axis,^8,9 with the more recent demonstration that reactive nitrogen intermediates derived from NO can post translationally modify protein thiol groups via S-nitrosylation/S-nitrosation.³⁹ It has recently been reported that circulating S-nitrosothiols are increased after acute NO₃⁻ ingestion and correlate with the reduction in brachial BP.⁴⁰ On the other hand, the effect of NO₃⁻ ingestion on plasma cGMP content is conflicting.^12,25,41 Further research is, therefore, required to provide insight into the potential biochemical mechanisms by which NO₃⁻ ingestion may elicit beneficial effects on cardiovascular function.

We aimed to investigate the dose–response relationship between NO₃⁻-rich BR (200, 400 and 800 mg NO₃⁻) and various markers of cardiovascular health and circulating biomarkers relating to NO₃⁻ metabolism and NO signalling compared to a control condition. We hypothesized that, following acute NO₃⁻ ingestion, plasma [NO₃⁻], [NO₂⁻], and [cGMP] would increase dose-dependently, while brachial and aortic BP, arterial stiffness (augmentation index and augmentation pressure) and macrovascular endothelial function would be improved following ingestion of 400 mg and 800 mg, but not 200 mg, NO₃⁻.

Methods

Participants

Seventeen healthy males completed this study (Table 1 and Fig. 1). All experiments were performed in accordance with the Human Tissue Act and UK Research and Innovation (UKRI) Guidelines, and procedures in this study were approved by the Loughborough University Research Ethics Approval Human Participants Sub Committee (ethics code: 14925). Participants provided written informed consent prior to study enrolment. None of the participants were smokers or vapers,⁴² nor did they use other dietary supplements or antibacterial mouthwash^43,44 from their familiarisation visit until study completion. Participants were normotensive, had no pre-existing health conditions, and were not taking any medications. Experimental trials occurred at the same time of day (±1 h) and participants refrained from high-intensity exercise and caffeine in the preceding 24 h and 6 h, respectively. Participants arrived at each trial 3 h post-prandial and replicated the same diet in the preceding 24 h. From familiarisation to completion of the study, participants were instructed to avoid overconsuming NO₃⁻-rich foods (beetroot, radishes, green beans, cress, celery, leafy green vegetables, processed meats) relative to their normal dietary intake of these foods.

Fig. 1 Flow diagram of participant progress through a crossover randomised controlled trial with three experimental conditions, including information on enrolment, allocation to study, follow-up, and data analysis.

Table 1 Baseline characteristics of seventeen healthy male participants

	Baseline
Age, height, body mass, body mass index (BMI), brachial systolic blood pressure (SBP), brachial diastolic blood pressure (DBP), heart rate (HR), and flow mediated dilation (FMD) at baseline, collected during the familiarisation visit. Data are presented as group mean ± SD.
Age (years)	23 ± 4
Height (m)	1.78 ± 0.80
Body mass (kg)	76 ± 12
BMI (kg m^−2)	24 ± 3
Brachial SBP (mmHg)	119 ± 4
Brachial DBP (mmHg)	65 ± 7
HR (bpm)	58 ± 9
FMD (%)	6.71 ± 4.72

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Study design

The study adopted a randomised, controlled, double-blind, crossover design, with three supplementation conditions providing 20 g of a formulated BR powder containing either 1% NO₃⁻ (200 mg), 2% NO₃⁻ (400 mg) or 4% NO₃⁻ (800 mg; TruBeet; Bio-gen Extracts Private Limited, Karnataka, India). Experimental visit order was counterbalanced across participants, and supplements were blinded to researchers and participants by someone who was not involved in any of the primary data collection by labelling the supplements in sealed opaque containers with ‘A’, ‘B’, or ‘C’, which were coded to a particular dose.

The 20 g supplement servings contained 13.33 g of formulated BR powder providing 1.5%, 3% and 6% NO₃⁻ in the 200 mg, 400 mg and 800 mg conditions, respectively. The remaining 6.67 g comprised of a sweetener (stevia), flavour (berry) and flavouring agents (malic & citric acid), included solely to improve palatability and participant compliance. The different BR powders were prepared by manipulating the processing control parameters (extraction time and temperature) during aqueous extraction to ensure precise NO₃⁻ concentration across the three different conditions, particularly for the low NO₃⁻grade material (200 mg). Specifically, raw beetroot was cleaned, shredded and extracted with water for 1 h at >90 °C with the residue obtained from the first extraction processed to obtain the low NO₃⁻ grade material. The aqueous extract was subsequently concentrated and spray-dried to obtain a free-flowing, water-soluble powder.

The NO₃⁻ content of BR powders were determined at two stages: first at the beetroot extraction stage and second after blending with flavouring agents and sweetener. NO₃⁻ quantification was performed on a dried basis using a colorimetric method where samples were treated with a salicylic acid-sulphuric acid mixture, and subsequently 2 N sodium hydroxide, before measuring absorbance using a UV-Vis spectrophotometer (UV-1800, Shimadzu, Japan) at 412 ± 2 nm with values compared to potassium NO₃⁻ standards.

All BR powders were mixed with 350 mL of water prior to ingestion. Participants attended the laboratory on four occasions, with visit one serving as a familiarisation session and visits two to four serving as the experimental testing sessions. Each visit was separated by at least a 48 h washout.

Familiarisation visit

After initially completing anthropometric measures, participants rested supine for 10 min before two brachial BP measures were completed using an automatic sphygmomanometer (Omron Healthcare, Kyoto, Japan). The FMD procedure was completed with information on the probe and cuff position in relation to anatomical landmarks, image depth, and image diameter recorded for replication in the subsequent experimental visits.

Experimental visits

Each experimental visit comprised a series of measurements assessed both prior to and following supplement ingestion. After 10 min supine rest in a dark, temperature controlled (22.2 ± 1.0 °C) laboratory, brachial BP, aortic BP and arterial stiffness variables, FMD assessments, and a venous blood sample were sequentially collected whilst the participant remained supine. Following this, the participant ingested the BR supplement followed by low NO₃⁻ water (Buxton mineral water; Buxton, UK) and two cereal bars (Nature Valley; General Mills, US). The participant underwent 2 h and 20 min of seated rest before returning to the laboratory for post-supplement assessments, to coincide with peak plasma [NO₂⁻] following acute NO₃⁻ ingestion.¹⁴ These assessments occurred in the same order as baseline, following a 10 min supine rest in a temperature and light-controlled laboratory.

Measurements

Brachial blood pressure. Brachial BP was assessed using an automatic sphygmomanometer (Omron Healthcare, Kyoto, Japan) on the left arm. BP was recorded 5 times over a 10 min period, while the participant was completely rested. The average of the 5 recordings was calculated and recorded.

Aortic blood pressure and arterial stiffness. An applanation tonometer (SphygmoCor; Atcor Medical, Sydney, Australia) was used to perform pulse wave analysis (PWA) assessed at the left radial artery. Mean brachial SBP and DBP from the prior recordings were used to calibrate the tonometer. A minimum of 2 satisfactory (>80% operator index) PWA recordings were recorded and averaged over 20 consecutive cardiac cycles. Data that failed to meet these criteria were discarded (n = 17 for CON, 200, and 400; n = 16 for 800). PWA variables of interest included: aortic SBP (mmHg), aortic DBP (mmHg), pulse rate (PR, bpm), augmentation pressure (AP, mmHg; the amplitude of the reflected wave), augmentation index (AIx, %; the relative reflected wave amplitude divided by pulse pressure and expressed as a percentage), and AIx adjusted for HR of 75 bpm (AIx@HR75).

Macrovascular endothelial function. Macrovascular endothelial function was assessed via FMD (following the assessment and analysis recommendations of Thijssen et al.⁴⁵ and Corretti et al.⁴⁶) in an air-conditioned room (20 °C) in response to a 5 min ischemic stimulus induced by forearm cuff inflation (SC5; Hokanson, Washington, USA). The participant lay supine with their right arm extended horizontally in line with the level of the heart. A manual straight segmental cuff was placed on the forearm, 3 cm distal and inferior to the cubital fossa and the brachial artery was located using a 9 L transducer at 9.0 MHz in an adjustable probe-holding device (LOGIQ E9 Ultrasound; GE Healthcare, Buckinghamshire, UK). Pulse-wave Doppler velocity was acquired simultaneously, with the insonation angle set at 60°. A baseline recording was taken for 1 min, before 5 min manual cuff inflation to 220 mmHg, followed by a final 3 min post occlusive reactive hyperaemia (PORH) recording to capture the peak arterial diameter. Recordings were collected via Vascular Imager (Medical Imaging Applications Vascular Research Tools, Iowa, USA) at a rate of 15 frames per second. An experienced sonographer conducted all FMD assessments (1.1% coefficient of variation for diameter, 11.5% coefficient of variation for FMD).

For all FMD analysis, an automated wall detection software package, Brachial Analyser, was used (Medical Imaging Applications Vascular Research Tools, Iowa, USA). The diameter parameters measured were mean brachial arterial diameter at baseline (BAD_ba, mm), peak brachial arterial diameter post-occlusion (BAD_pk, mm), time to BAD_pk (TTP, s), absolute change in brachial arterial diameter (BAD_FMD, mm), and relative change in brachial arterial diameter (FMD, %). BAD_ba was calculated as the mean value over 30 s. For BAD_pk, the three greatest diameters were found, and 3 s averages were calculated. The highest of the three outputs was taken as BAD_pk. The time aligned to this point was recorded as the TTP.

The equations for BAD_FMD and FMD were:

BAD_FMD (mm) = BAD_pk − BAD_ba

FMD (%) = ((BAD_pk − BAD_ba)/BAD_ba) × 100.

Shear rate (SR) was calculated, via the equation below, at baseline (SR_base; reported as the average over 30 s) and PORH (SR_AUC; reported as the sum of the area under the curve from cuff release to TTP). FMD (%) was normalised to SR_AUC (FMD_SR) via the following equations:

SR (s⁻¹) = 4 × (mean blood flow velocity/BAD)

FMD_SR (% s⁻¹) = FMD/SR

Venous blood collection and analysis. Venous blood samples were taken from the antecubital-fossa region, acquiring samples in lithium heparin (LH), ethylenediaminetetraacetic acid (EDTA), and serum vacutainers. All samples were collected (n = 17 for CON, 200 mg and 800 mg) except one post 400 mg NO₃⁻ (n = 16). Samples were centrifuged at 3500g for 5 min to obtain LH plasma, or 1500g for 15 min to obtain EDTA plasma and serum. Samples were immediately stored at −80 °C for subsequent analysis.

Plasma [NO₂⁻] and [NO₃⁻] were analysed using LH plasma via ozone chemiluminescence. This process is described in full in previous work.²⁰ In brief, this method of quantification involved deproteinising plasma samples with ethanol prior to subsequent [NO₃⁻] analysis. 50 μL deproteinised samples were injected into a purge vessel containing 0.8% (w/v) vanadium chloride in 1 M HCl for determination of plasma [NO₃⁻], via an ozone-based chemiluminescence NO analyser (Sievers NOA 280i, Analytix Ltd, Durham, UK), by its reduction to NO. For [NO₂⁻] determination, plasma samples were deproteinised in ethanol and 200 μL of the supernatant was injected into the purge vessel prepared with glacial acetic acid and aqueous sodium iodide (4% w/v) to quantify plasma [NO₂⁻] by its reduction to NO. Plasma [NO₃⁻] and [NO₂⁻] were determined by plotting signal area (mV) against calibration plots of NaNO₃ and NaNO₂ standards, respectively. Origin Lab was used to smooth the signal and objectively identify the peaks.

Plasma [cGMP] was measured using EDTA plasma via a competitive commercial assay (Cyclic GMP ELISA kit; Cayman Chemical, Michigan, USA).

Statistical analysis

Sample size was estimated with a priori power analysis for changes in FMD using G*Power (G*Power 3.1.97; Kiel, Germany). The analysis suggested with a change in means and SD of 1% and 1, respectively, and an assumption of 80% power and a significance level of 0.05, the total number of participants required to detect a statistically significant change in FMD was 17 (large effect size; η_p² = 0.20). The mean for the analysis was estimated from similarly designed studies based on a 1% change in FMD being clinically meaningful.^45,47 This is higher than the sample sizes in previous similar studies assessing the effects of a single dose of beetroot juice on cardiovascular function in healthy males that detected statistically significant changes in FMD with 10–11 participants.^27,30 Statistical analysis was performed using Jamovi (version 2.3.28; Jamovi, Sydney, Australia). Visual inspection of Q–Q plots and residual histograms were used to confirm data normality.⁴⁸ One-way linear mixed models (LMM) were performed for all variables to account for missing data points. BAD_ba was included as a covariate for LMM analysis of FMD. Four conditions were compared: control (CON, taken as the average of the three experimental visit baseline results), 200 mg NO₃⁻, 400 mg NO₃⁻, 800 mg NO₃⁻. Significant main condition effects were analysed further using Holm–Bonferroni corrected paired t tests. To calculate effect sizes, partial eta squared (n_p²) was used for the omnibus tests, where effects were classified as small (n_p² ≥ 0.01), medium (n_p² ≥ 0.06), or large (n_p² ≥ 0.14).⁴⁹ Hedges g (g) was used for paired-sample's t tests, where effects were classified as (g ≥ 0.2), medium (g ≥ 0.5), or large (g ≥ 0.8).⁴⁹ All data are displayed as mean ± SD unless stated otherwise. Statistical significance was accepted at P < 0.05. All variables for analysis had a sample size of 17.

Results

Plasma [NO₃⁻], [NO₂⁻], and [cGMP]

There was a main effect of condition for plasma [NO₃⁻] (P < 0.001, n_p² = 0.973). Post-hoc analyses revealed a significant dose-dependent increase in plasma [NO₃⁻] (CON: 50 ± 16 μM, 200: 99 ± 26 μM, 400: 293 ± 42 μM, 800: 482 ± 74 μM; P < 0.001, g = 2.216–7.879). This represents 2-fold, 6-fold, and 10-fold increases in plasma [NO₃⁻] following 200, 400, and 800 conditions, respectively. There was a main effect of condition for plasma [NO₂⁻] (P < 0.001, n_p² = 0.681), with plasma [NO₂⁻] increasing dose-dependently following 400 mg (300 ± 114 nM) and 800 mg NO₃⁻ (423 ± 228 nM; P < 0.008, g = 0.666–2.714). This represents a 4-fold and 6-fold increase in plasma [NO₂⁻] following 400 and 800 conditions, respectively. Plasma [NO₂⁻] was not significantly different between CON (71 ± 24 nM) and 200 mg NO₃⁻ (103 ± 48 nM), though the effect size was large (P = 0.433, g = 0.823). There was no main effect of condition for plasma [cGMP] (P = 0.087, n_p² = 0.127; CON: 4.9 ± 1.5 pmol mL⁻¹, 200: 5.4 ± 2.9 pmol mL⁻¹, 400: 4.3 ± 1.9 pmol mL⁻¹, 800: 4.6 ± 1.4 pmol mL⁻¹; Fig. 2).

Fig. 2 Plasma nitrate ([NO₃⁻]), nitrite ([NO₂⁻]), and cyclic guanosine monophosphate ([cGMP]) concentrations following four conditions: no supplement (CON), and NO₃⁻-rich beetroot powder providing 200 mg (200), 400 mg (400) or 800 mg (800) NO₃⁻. The bars represent the group mean ± SD responses with the filled circles representing individual participants. * denotes significantly different from CON (P < 0.05). ^# denotes significantly different from 200 (P < 0.05). † denotes significantly different from 400 (P < 0.05).

Macrovascular endothelial function

Brachial artery diameter. There were no significant differences in BAD_ba or BAD_pk between conditions (P = 0.089–0.889, n_p² = 0.013–0.126; Table 2). There was a significant main effect of condition for TTP (P = 0.016, n_p² = 0.192; Table 2). Post-hoc tests revealed a significant reduction in TTP following 400 (P = 0.017, g = 1.129), but not 200 (P = 0.074, g = 1.09) or 800 (P = 0.430, g = 0.376), compared to CON (Table 2).

Table 2 Markers of macrovascular endothelial function assessed following four doses of nitrate-rich beetroot juice

	CON	200	400	800
Brachial artery diameter at baseline (BADba) and peak (BADpk), time to peak diameter (TTP), absolute change in brachial artery diameter (BADFMD), relative flow mediated dilation (FMD), shear rate at baseline (SRbase), accumulative shear rate from post occlusion to TTP (SRAUC), and flow mediated dilation normalised to shear rate (FMDSR) following four conditions: no supplementation (CON; n = 17), and NO3^−-rich beetroot powder providing 200 mg (200; n = 17), 400 mg (400; n = 17) or 800 mg (800; n = 16) NO3^−. Data are presented as group mean ± SD. * denotes different from CON (P < 0.05). ^# denotes significantly different from 200 (P < 0.05).
BADba (mm)	4.09 ± 0.41	4.13 ± 0.46	4.13 ± 0.46	4.13 ± 0.48
BADpk (mm)	4.32 ± 0.42	4.37 ± 0.45	4.48 ± 0.50	4.41 ± 0.53
TTP (s)	49 ± 9	40 ± 7	38 ± 10*	44 ± 16
BADFMD (mm)	0.23 ± 0.09	0.24 ± 0.11	0.35 ± 0.23*	0.28 ± 0.15
FMD (%)	5.61 ± 2.26	5.99 ± 2.84	8.68 ± 5.71*	6.66 ± 3.69
SRbase (s^−1)	19.43 ± 6.83	14.03 ± 4.66	14.76 ± 6.60	16.76 ± 8.86
SRAUC (s^−1)	2775 ± 838	2589 ± 610	2394 ± 865	2610 ± 906
FMDSR (% s^−1)	2.15 ± 1.08	2.23 ± 1.05	4.31 ± 3.51*^#	2.57 ± 1.64

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Flow mediated dilation. There were significant main effects for condition for BAD_FMD (P = 0.027, n_p² = 0.172) and FMD (P = 0.037, n_p² = 0.161). Post-hoc tests revealed a greater BAD_FMD (P = 0.036, g = 0.671) and FMD (P = 0.047, g = 0.690) in 400 mg compared to CON (+0.12 mm and +3.07%; Table 2, Fig. 3). Following 200 mg and 800 mg, BAD_FMD (P = 0.816–0.860, g = 0.097–0.395) and FMD (P = 1.000, g = 0.145–0.335) were not different from CON (200: +0.01 mm and +0.38%, 800: +0.05 mm and +1.05%; Table 2, Fig. 3). There remained a significant main effect for FMD when accounting for BAD_ba as a covariate (P = 0.025, n_p² = 0.182), where FMD was greater in 400 mg (P = 0.029, g = 0.690), but not 200 mg (P = 1.000, g = 0.145) or 800 mg (P = 0.858, g = 0.335) compared to CON. There was a main effect of condition for FMD_SR (P = 0.011, n_p² = 0.223). Post-hoc analyses revealed greater FMD_SR in 400 mg compared to CON (P = 0.020, g = 0.812) and 200 mg (P = 0.022, g = 0.784), but no other between-condition differences (P = 0.081–1.000, g = 0.073–0.620; Table 2).

Fig. 3 Absolute change in brachial artery diameter (BAD_FMD) and relative flow mediated diameter (FMD) following four conditions: no supplement (CON), and NO₃⁻-rich beetroot powder providing 200 mg (200), 400 mg (400) or 800 mg (800) NO₃⁻. Bars represent the group mean ± SD responses with the filled circles representing individual participants. * denotes different from CON (P < 0.05).

Aortic blood pressure and arterial stiffness

Aortic blood pressure. There was a significant main effect of condition for aortic SBP (P = 0.025, n_p² = 0.178) and post-hoc tests revealed a significant reduction in aortic SBP in 800 mg (−4 mmHg; P = 0.032, g = 0.976), but not 200 mg (−3 mmHg; P = 0.119, g = 0.514) or 400 mg (−2 mmHg; P = 0.681, g = 0.431), in comparison to CON (Table 3). There was a main effect of condition for PR (P = 0.043, n_p² = 0.157), however post-hoc tests revealed no significant between-condition differences (P = 0.068–0.682, g = 0.108–0.434; Table 3). There was no main effect of condition for aortic DBP (P = 0.374, n_p² = 0.063; Table 3).

Table 3 Aortic blood pressure and arterial stiffness variables following four doses of nitrate-rich beetroot juice

	CON	200	400	800
Aortic systolic blood pressure (SBP), aortic diastolic blood pressure (DBP), augmentation pressure (AP), augmentation index (AIx), and augmentation index relative to heart rate (AIx@75HR) following four conditions: no supplementation (CON), and NO3^−-rich beetroot powder providing 200 mg (200), 400 mg (400) or 800 mg (800) NO3^−. Data are presented as group mean ± SD. * denotes different from CON (P < 0.05).
SBP (mmHg)	99 ± 4	96 ± 7	97 ± 5	95 ± 4*
DBP (mmHg)	65 ± 7	64 ± 9	65 ± 7	63 ± 9
PR (bpm)	57 ± 9	54 ± 9	55 ± 6	53 ± 9
AP (mmHg)	1 ± 3	0 ± 3	−1 ± 4*	−1 ± 4*
AIx (%)	3 ± 10	−1 ± 10	−3 ± 13*	−3 ± 12*
AIx@75HR (%)	−5 ± 8	−11 ± 8*	−12 ± 12*	−13 ± 12*

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Arterial stiffness variables. There were significant main effects of condition for AIx (P = 0.006, n_p² = 0.230), AIx@75HR (P = 0.003, n_p² = 0.259) and AP (P = 0.008, n_p² = 0.221; Table 3). Post-hoc analyses revealed lower AIx and AP in 400 mg (−6% and −2 mmHg; P = 0.010–0.017, g = 0.505–0.552) and 800 mg (−6% and −2 mmHg; P = 0.016–0.019, g = 0.532–0.554), but not 200 mg (−4% and −1 mmHg; P = 0.051–0.114, g = 0.325–0.391), compared to CON (Table 3). AIx@75HR was significantly lower than CON in 200 mg (−6%; P = 0.047, g = 0.732), 400 mg (−7%; P = 0.006, g = 0.670), and 800 mg (−8% P = 0.006, g = 0.770; Table 3). There was no difference in AIx, AIx@75HR, or AP between 200 mg, 400 mg, and 800 mg conditions (P = 0.854–1.000, g = 0.000–0.505; Table 3).

Brachial artery blood pressure

There were no differences in brachial SBP or DBP between conditions (P = 0.151–0.500, n_p² = 0.048–0.104; Table 4). There was a significant main effect of condition for HR (P = 0.022, n_p² = 0.180). Post-hoc tests revealed that HR was significantly lower in 200 mg (−4 bpm; P = 0.032, g = 0.434), but not 400 mg (−2 bpm; P = 0.573, g = 0.255) or 800 mg (−3 bpm; P = 0.062, g = 0.325), compared to CON (Table 4).

Table 4 Brachial blood pressure and heart rate following four doses of nitrate-rich beetroot juice (n = 17)

	CON	200	400	800
Brachial systolic blood pressure (SBP), brachial diastolic blood pressure (DBP), and heart rate (HR) following four conditions: no supplementation (CON), and NO3^−-rich beetroot powder providing 200 mg (200), 400 mg (400) or 800 mg (800) NO3^−. Data are presented as group mean ± SD. * denotes different from CON (P < 0.05).
SBP (mmHg)	118 ± 5	116 ± 7	117 ± 4	115 ± 5
DBP (mmHg)	65 ± 7	64 ± 9	65 ± 7	63 ± 7
HR (bpm)	58 ± 9	54 ± 9*	56 ± 6	55 ± 9

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Discussion

The primary original findings of this study were that, compared to CON: (1) macrovascular endothelial function was improved after ingesting 400 mg NO₃⁻, but not 200 mg or 800 mg NO₃⁻; (2) all NO₃⁻ doses improved arterial stiffness variables and this improvement was not dose-dependent; (3) aortic SBP was decreased after ingesting 800 mg NO₃⁻, but not 200 mg or 400 mg NO₃⁻; and (4) plasma [cGMP] was unaffected following ingestion of any of the NO₃⁻ doses administered. These findings improve understanding of the effects of NO₃⁻ supplementation on cardiovascular function in healthy, normotensive, young adults by revealing differing dose–response effects on established cardiovascular health markers.

Cardiovascular function

The dose–response relationship between NO₃⁻ supplementation and BP has been examined previously;^12,14 however, no such investigation had been conducted for NO-mediated macrovascular endothelial function. In the current study, BAD_FMD and FMD increased by 0.12 mm and 3%, respectively, after ingesting 400 mg NO₃⁻ compared to CON. In contrast, neither BAD_FMD nor FMD were different between the CON, 200 mg NO₃⁻ and 800 mg NO₃⁻ conditions. Consistent with the clinically meaningful increase in FMD following 400 mg NO₃⁻ in comparison to CON, Yuschen et al.³⁰ and Bakker et al.²⁷ observed 2% increases in FMD following ingestion of 310–400 mg NO₃⁻ in healthy young adults. Some other studies have reported increased FMD following ingestion of a larger NO₃⁻ dose (∼600–800 mg) in older adults and pregnant females.^28,50–52 It is well established that older age and lower concentration of oestrogen negatively impact macrovascular endothelial function,^51,53 which could account for some of these inter-study differences in the NO₃⁻ dose required to improve macrovascular endothelial function. Interestingly, Bahra et al.³¹ observed no change in FMD following ingestion of ∼500 mg KNO₃ in young healthy adults, which may be linked to other potential active nutrients in beetroot, such as (poly)phenols, or betalains, that could have contributed to the increase in macrovascular endothelial function observed in the present study.^16,32,54

The beneficial effects of NO₃⁻ supplementation on the cardiovascular system have been largely ascribed to the increase in plasma [NO₂⁻] and its subsequent potential to either directly nitrosylate/nitrosate protein thiols groups, or to undergo a one-electron reduction to NO.⁶ The lack of change in macrovascular endothelial function after ingesting 200 mg NO₃⁻ in the current study may be a function of an insufficient increase in plasma [NO₂⁻]. Indeed, it has been suggested that plasma [NO₂⁻] may need to increase above a certain threshold concentration, perhaps 200 nM, before beneficial effects are observed.⁵⁵ Consistent with this postulate, plasma [NO₂⁻] was 103 ± 48 nM after ingesting 200 mg NO₃⁻ and macrovascular endothelial function was not improved, whereas macrovascular endothelial function was improved after ingesting 400 mg NO₃⁻ when plasma [NO₂⁻] was 300 ± 114 nM. However, despite a further increase in plasma [NO₂⁻] to 423 ± 228 nM after ingesting 800 mg NO₃⁻, macrovascular endothelial function was not improved compared to CON. This curtailment in the beneficial effects of NO₃⁻ dose on endothelial function may be a function of crosstalk between NO derived from endothelial NOS (eNOS) and the NO₃⁻–NO₂⁻–NO pathway.⁵⁶ The FMD technique assesses NO-mediated endothelial function in response to shear stress and the subsequent activation of eNOS.^1,45 However, during the occlusion that precedes the FMD assessment, the decline in brachial artery PO₂ and pH would be expected to aid the reduction of NO₂⁻ to NO.⁵⁷ This potential for increased NO₂⁻-derived NO after ingestion of 400 mg NO₃⁻ may have complemented and augmented eNOS-derived NO to increase FMD. However, it is also recognised that elevated NO can interfere with eNOS activity via S-nitrosylation.⁵⁸ It is possible, therefore, that the higher plasma [NO₂⁻] after ingesting 800 mg NO₃⁻ may have increased NO₂⁻-derived NO to a greater extent than after ingesting 400 mg NO₃⁻ which could have impeded eNOS-derived NO such that the net NO generation may have been lower.

Arterial stiffness variables were the most responsive to ingestion of NO₃⁻-rich BR in the current study, and in contrast to the effects on endothelial function, all NO₃⁻ doses administered improved AIx@75HR by a similar magnitude, whilst AIx and AP were only improved following 400 mg and 800 mg NO₃⁻. Lower AIx has previously been reported following acute ingestion of 400–600 mg NO₃⁻, in both low- and high-CVD risk populations^21,36,59 suggesting that NO₃⁻-rich BR has the potential to lower arterial stiffness. Consistent with a greater potential for acute NO₃⁻-rich BR supplementation to improve arterial stiffness than endothelial function markers in the current study, a previous study reported improved arterial stiffness but not FMD after acute NO₃⁻ ingestion in healthy adults.³¹ The physiological bases of AIx and AP are not entirely clear,⁶⁰ which complicates interpretation of the putative physiological mechanisms that could underpin improved AIx and AP after NO₃⁻-rich BR supplementation in the current study. Conventionally, AIx and AP have been principally attributed to the timing of reflected waves from the periphery arriving at the aorta, with earlier arrival during systole resulting in higher values, and delayed arrival during diastole resulting in lower values.⁶¹ As such, it is possible that greater potential for NO-induced vasodilation after NO₃⁻-rich BR supplementation could have mediated the improvements (lowering) in AIx and AP in the current study by increasing arterial compliance, leading to slowing of wave reflections from the periphery, delaying their arrival at the heart.³⁶ More recently, it has been suggested that AIx and AP are more closely mediated by an aortic reserve reservoir⁶² such that NO₃⁻-rich BR supplementation may have improved aortic compliance and distensibility through increased NO signalling. An interesting and unexpected observation from the current study was improved AIx@75HR after ingestion of 200 mg NO₃⁻. In contrast, previous research^24,34,63 found no improvements in pulse wave variables in healthy adults following the administration of low doses of NO₃⁻ (70–300 mg). However, since NO₃⁻ was administered in the form of whole food diets, as opposed to controlled NO₃⁻-rich BR supplements, and given that the NO₃⁻ supplementation vehicle can influence NO₃⁻ metabolism and cardiovascular responses,^16,32,33 this could account for the inter-study differences. Since AIx@75HR was improved with all BR doses administered in the current study, it should also be acknowledged that some other nutrients in the BR powder, such as antioxidants or polyphenols,⁶⁴ could have, independently, or synergistically with NO₃⁻, contributed to the improvements in arterial stiffness markers in the current study.^16,32,65 Therefore, whilst NO₃⁻-rich BR supplementation appeared to be particularly potent at improving arterial stiffness markers, the mechanisms that underlie such effects are not entirely clear.

In the current study, aortic SBP was lowered following 800 mg NO₃⁻, but 200 mg or 400 mg NO₃⁻. This finding is consistent with results from Rowland et al.²⁰ who reported a reduction in aortic SBP in healthy males following acute ingestion of a ∼832 mg dietary NO₃⁻-rich BR dose. However, despite previous reports of a lowering in brachial BP following acute dietary NO₃⁻ ingestion in healthy participants^{12,20,22,29,34,66} no changes in brachial SBP or DBP were observed following any dose of NO₃⁻-rich BR in the present study. Nonetheless, the findings of the current study are consistent with some previous research.^2,30,50 Since the participants in the current study were normotensive and that the efficacy of NO₃⁻ supplementation to lower BP appears to diminish with a progressively lower baseline BP,^12,18 this could account for the lack of an effect of NO₃⁻-rich BR supplementation on brachial BP in the current study.

Dietary nitrate metabolism and NO signalling

In agreement with previous research, plasma [NO₃⁻] increased dose-dependently following the acute ingestion of 200 mg, 400 mg, and 800 mg NO₃⁻-rich BR in the present study.^12,14 However, and in contrast with previous findings,¹⁴ the increase in plasma [NO₂⁻] was not dose-dependent, with no significant increase following ingestion of BR providing 200 mg NO₃⁻. Nonetheless, plasma [NO₂⁻] was increased dose-dependently following ingestion of 400 and 800 mg NO₃⁻, consistent with previous observations.¹⁴ Previous research investigating low-dose NO₃⁻ supplementation via the administration of a NO₃⁻-rich diet, did not investigate plasma [NO₂⁻] response,³⁴ or observed small increases (1-fold) in plasma [NO₂⁻].²⁴ Previous multiple-dose studies have failed to investigate the plasma NO₂⁻ response in doses as low as the present study.^12,14 Moreover, although there was not a statistically significant increase in plasma [NO₂⁻] between 200 mg NO₃⁻ and CON, the effect size was large, and this was accompanied by improved arterial stiffness. Therefore, a NO₃⁻ dose as low as 200 mg may be sufficient to improve aspects of cardiovascular function such that the increase in plasma [NO₂⁻] elicited by this NO₃⁻ dose may be clinically relevant. Alternatively, some of the effects of NO₃⁻ and NO₂⁻ administration might not be driven entirely by NO₂⁻.^15,67 The absence of a change in plasma [cGMP] following any dose of NO₃⁻-rich BR in the current study implies that vascular improvements were not cGMP-mediated. This agrees with previous work that observed no change in plasma [cGMP] following acute supplementation with 7 mg kg⁻¹ potassium NO₃⁻ in young and older adults with obesity.⁴¹ Conversely, Kapil et al.¹² observed significant elevations in plasma [cGMP] 3 h post-supplementation, however, the supplement form and dose administered differed to those in the current study (1488 mg KNO₃), potentially accounting for these disparate effects.

Potential implications

The findings of the present study suggest that acute ingestion of a 200–800 mg NO₃⁻ dose can improve markers of arterial stiffness whilst a 400 mg dose can improve endothelial function in healthy young adults. Endothelial function is a well-established risk factor for CVD, with an FMD <5% categorised as impaired.^1,45 Meta-analyses have highlighted that a 1% increase in FMD conferred an 8–13% reduction in risk of CVD independent of conventional CVD risk factors.^53,68 Therefore, the 3% mean improvement in FMD in the current study after ingesting 400 mg NO₃⁻ implies a potentially clinically relevant reduction in CVD morbidity in young healthy males. Moreover, in healthy adults a 1-point standard deviation increase in AIx translates to a 23% rise in risk of cardiovascular mortality⁶⁹ and a 10 mmHg reduction in AP translates to a 20% reduction in CVD development.⁷⁰ Therefore, the results of the current study imply a potential acute reduction in risk of cardiovascular mortality. Collectively, the current findings suggest that acute ingestion of a BR supplement providing 400 mg NO₃⁻ can elicit clinically relevant changes in arterial stiffness and endothelial function in support of the potential cardioprotective effects of BR supplementation, at least in healthy young males. Furthermore, since pressures change throughout the arterial tree, brachial BP is not a true reflection of aortic BP and emerging evidence suggests that aortic BP is more predictive of future cardiovascular events than brachial BP.⁷¹ Thus, the current findings provide some evidence to support that acute ingestion of 800 mg NO₃⁻-rich BR may be cardioprotective.

Experimental considerations

The principal limitation of the current study was that production difficulties with obtaining the low NO₃⁻ grade BR for a placebo resulted in this study not adopting a placebo-controlled design. Instead, the three NO₃⁻-rich BR doses were compared to a pre-ingestion CON value. A further potential limitation of this approach is that pre and post BR ingestion assessments were separated by at least 2.5 h, which could have been confounded by potential circadian effects of the outcome variables. However, this concern is somewhat mitigated by a recent paper suggesting that the efficacy of BR to improve cardiovascular function appears to be consistent across the day with minimal circadian effects on BP and arterial stiffness variables.²⁰ Whilst the lack of any inter-condition differences in plasma [cGMP] following BR implies that vascular improvements were not cGMP-mediated, since [cGMP] was assessed in venous plasma, it is not possible to exclude the possibility that cGMP may have been elevated in different components of the vascular tree and may have contributed to some of the beneficial cardiovascular effects observed after NO₃⁻ ingestion in the current study. It has recently been suggested that the increase in whole blood [RSNO] following BR consumption is inversely correlated with the reduction in BP.¹⁵ Therefore, the lack of assessment of blood [RSNO] in the current study is recognised as a limitation.

Conclusions

In young healthy males, acute ingestion of BR providing 400 mg NO₃⁻ improved macrovascular endothelial function, with arterial stiffness variables comparably improved after ingestion of 200–800 mg NO₃⁻. Furthermore, aortic SBP was improved exclusively after ingesting 800 mg NO₃⁻. There were no improvements in brachial BP or plasma [cGMP] after ingesting 200–800 mg NO₃⁻. These findings improve our understanding of the effect of BR supplementation on risk factors for CVD and suggest that a single BR dose providing 400 mg NO₃⁻ can elicit clinically relevant improvements in endothelial function and arterial stiffness. While long-term studies are needed to confirm sustained benefits, these data lend further support to the notion that a diet rich in NO₃⁻-containing vegetables may confer some cardiovascular health benefits.

Author contributions

AGM: conceptualization, formal analysis, investigation, methodology, project administration, resources, visualization, writing – original draft. EOD: methodology, supervision, writing – review & editing. JPA: investigation, writing – review & editing. SNR: methodology, supervision, writing – review & editing. AIS: methodology, validation, writing – review & editing. MP: methodology, validation, writing – review & editing. LJJ: supervision, writing – review & editing. TC: validation, supervision, writing – review & editing. SJB: conceptualization, funding acquisition, methodology, supervision, writing – review & editing.

Conflicts of interest

The authors declare no conflicts of interest regarding this manuscript.

Data availability

The data for this article are available from Loughborough University Research Repository (https://repository.lboro.ac.uk/) at [URL – https://doi.org/10.17028/rd.lboro.30768083].

Acknowledgements

This research was financially supported by a match-funded PhD studentship from Loughborough University and Bio-Gen Extracts (Ltd). Bio-Gen Extracts (Ltd) played no role in the study design, data analysis or interpretation, or the writing of this manuscript. This research was carried out at the National Institute for Health and Care Research (NIHR) Leicester Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.

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