Abstract
Aims
To test if spironolactone or dietary nitrate from beetroot juice could reduce arterial stiffness as aortic pulse wave velocity (PWVart), a potential treatment target, independently of blood pressure.
Methods
Daily spironolactone (≤50 mg) vs doxazosin (control ≤16 mg) and 70 mL beetroot juice (Beet‐It ≤11 mmol nitrate) vs nitrate‐depleted juice (placebo; 0 mmol nitrate) were tested in people at risk or with type‐2 diabetes using a double‐blind, 6‐month factorial trial. Vascular indices (baseline, 12, 24 weeks) were cardiac–ankle vascular index (CAVI), a nominally pressure‐independent stiffness measure (primary outcome), PWVart secondary, central systolic pressure and augmentation. Analysis was intention‐to‐treat, adjusted for systolic pressure differences between trial arms.
Results
Spironolactone did not reduce stiffness, with evidence for reduced CAVI on doxazosin rather than spironolactone (mean difference [95% confidence interval]; 0.25 [−0.3, 0.5] units, P = .080), firmer for PWVart (0.37 [0.01, 0.7] m/s, P = .045). There was no difference in systolic pressure reduction between spironolactone and doxazosin (0.7 [−4.8, 3.3] mmHg, P = .7). Circulating nitrate and nitrite increased on active vs placebo juice, with central systolic pressure lowered −2.6 [−4.5, − 0.8] mmHg, P = .007 more on the active juice, but did not reduce CAVI, PWVart or peripheral pressure. Change in nitrate and nitrite concentrations were 1.5‐fold [1.1–2.2] and 2.2‐fold [1.3, 3.6] higher on spironolactone than on doxazosin respectively; both P < .05.
Conclusion
Contrary to our hypothesis, in at‐risk/type 2 diabetes patients, spironolactone did not reduce arterial stiffness, rather PWVart was lower on doxazosin. Dietary nitrate elevated plasma nitrite, selectively lowering central systolic pressure, observed previously for nitrite.
Keywords: arterial stiffness, beetroot juice, blood pressure, dietary nitrate, nitrate–nitrite–NO, pathway, type 2 diabetes
What is already known about this subject
Arterial stiffness is a predictor of mortality, independently of blood pressure (BP) and diabetes.
Inorganic dietary nitrate has been shown to reduce blood pressure and arterial stiffness via the nitrate–nitrite nitric oxide pathway.
Spironolactone is reported to reduce arterial stiffness, but if this is BP‐independent is not clear.
What this study adds
The longest trial to test inorganic nitrate on vascular parameters to date.
Inorganic dietary nitrate selectively reduced central systolic BP which parallels previous data.
Despite lowering BP slightly more than did the α‐blocker, doxazosin, spironolactone did not reduce arterial stiffness, which was marginally lowered on doxazosin.
1. INTRODUCTION
Type 2 diabetes mellitus (T2DM) is characterised by excess cardiac and vascular disease even before formal diagnosis.1, 2 Arterial stiffness measured as aortic pulse wave velocity (PWVart) is amongst the most powerful predictors of both cardiovascular and all‐cause mortality, crucially independent of mean or systolic blood pressure (SBP) and other standard risk factors, including glycaemia.3 Reducing arterial stiffness could be particularly valuable in overweight people at increased risk of or already with overt T2DM, because of its predictive impact in glucose intolerance/T2DM,4 and its high prevalence in these people since early measures of arterial stiffness were used.5, 6, 7 The pathology of arterial stiffness involves elastin degradation and collagen deposition with fibrosis from inflammatory stimuli including dysregulation of nitric oxide (NO)8 and upregulation of profibrotic factors.9, 10, 11
Reductions in PWV by lifestyle measures are reported particularly for exercise, weight loss and specific dietary components, and by various pharmacological agents, including antihypertensives, statins, some antidiabetic medications and advanced glycation end‐product breakers.12 However, PWV reduction formally independent of BP is seldom examined. Doing so is important as PWV is intrinsically linked to BP hence it can be hard to distinguish the two.
Spironolactone, a mineralocorticoid receptor antagonist was recently found highly effective in reducing BP in proven resistant hypertension.13 Initial trials for specifically reducing arterial stiffness, in early kidney disease,14 untreated hypertensives15 and dilated cardiomyopathy16 appeared promising. These trials were generally not designed to test impact on PWV formally independent of BP change.
Inorganic (dietary) nitrate, abundant in green leafy vegetables and beetroot17 reduces BP in healthy18 and hypertensive volunteers19 via the nitrate–nitrite–NO pathway,20 but not in patients with T2DM,21, 22 or with their inclusion in a meta‐analysis of 24 h ambulatory BP monitoring.23 PWV reductions with inorganic nitrate have also been noted in healthy and hypertensive volunteers, but over too short a period for vessel remodelling; these were likely BP‐dependent reductions.24 We found that inorganic nitrite selectively lowers aortic, relative to peripheral, BP, with reductions also in PWV that seem to be via selective normoxia‐dependent conduit (radial) artery dilatation in healthy volunteers,25, 26 and selectively dilated epicardial coronary arteries in patients undergoing coronary angiography.27 While tolerance develops to organic nitrates,28 it has not been described for inorganic nitrate,19 perhaps this due to the mechanisms of bioactivation of inorganic nitrite to nitric oxide, and suppression of reactive oxygen species.29 Longer‐term effects of inorganic nitrate beyond 6 weeks have not yet been tested.
In the trial reported here, we hypothesised that spironolactone and dietary nitrate would reduce arterial stiffness independently of BP reduction in people with or at risk of T2DM. We tested this hypothesis in a double‐blind, controlled, factorial design 24‐week trial using cardiac–ankle vascular index (CAVI) as the primary measure of stiffness and PWVart adjusted for BP change as the secondary outcome.
2. METHODS
2.1. Study design and interventions
A single centre, double‐blind, parallel, randomised controlled intervention trial in a 2 × 2 factorial design was carried out in accordance with the Declaration of Helsinki and US Code of Federal Regulations.
Participants were assigned to 1 of 4 arms using computer randomization in blocks of 6, by an independent party. Interventions were spironolactone (12.5 mg daily for 1 week, 12.5 mg twice daily for 11 weeks, increased to 25 mg twice daily to 24 weeks) with doxazosin as its control (4 mg similarly titrated to 8 mg twice daily) and dietary nitrate as beetroot juice (7.5 mmol nitrate increased at 12 weeks to 11.2 mmol nitrate, as measured in our laboratory) or nitrate‐free beetroot juice as placebo (0 mmol nitrate; see Supplementary texthttp://hyper.ahajournals.org). Spironolactone and doxazosin were prepared in indistinguishable brown bottles by St Thomas' Hospital pharmacy, London, UK. Commercially available beetroot juice, ‘Beet It' and ‘Beet It SPORT' were supplied as 15 × 70‐mL bottles, indistinguishable between active and control juice, prepared and supplied by James White Drinks, Ltd, Suffolk UK.
Participants with or at risk of T2DM were recruited from Guy's and St Thomas' Hospitals, London, UK and surrounding areas between 2013–2015. Inclusion criteria were age 18–80 years, clinically diagnosed T2DM or at risk of T2DM (as body mass index ≥27 kg/m2, positive family history or glucose intolerance after 75 g challenge), ability to understand and comply with the protocol. Exclusion criteria: interfering chronic illness, adverse reaction to either drug, known allergy to beetroot, estimated glomerular filtration rate <45 mL min−1, HbA1c >11% (97 mM/M), pregnant, breast feeding or atrial fibrillation. Written informed consent was obtained from all participants. The protocol was approved by South London Research Ethics Committee.
The primary outcome was change in arterial stiffness, nominally independent of BP, as measured by CAVI. Secondary outcomes were arterial stiffness, as measured by PWVart, with central BP and augmentation index. Both primary and secondary outcomes were to be adjusted for differences in peripheral baseline BP and BP change between trial arms at start and finish.
At St Thomas' Hospital Clinical Research Facility, London, participants rested supine in a temperature‐controlled room for 20 minutes. Vascular measures were then performed supine in random order according to institutional guidelines.
After anthropometry, CAVI was measured using the VS‐1500 N, VaSera machine (Fukuda Denshi Ltd, Japan) as described.30 Microphone‐detected heart sounds were monitored, with BP cuffs on each arm and above each ankle, with pulse waves detected by the cuffs at 30–50 mmHg. CAVI was calculated from PWV, as pulse wave transit times from aortic valve (second sound) to ankle: CAVI = [ln SBP/ln DBP]. [2ρ/ΔP]. PWV2, with path length estimated from height.31 CAVI was measured in duplicate and averaged. CAVI0 data were calculated as described previously.32 PWVart, peripheral systolic, diastolic and central BP, aortic and brachial augmentation index and heart rate from 6–8 cardiac cycles were measured using appropriately sized cuffs by Arteriograph 24 device (TensioMed Kft. Hungary), analysing mean of duplicate good quality readings. Quality was prespecified with Arteriograph and VaSera waveforms checked by the manufacturers, blinded to other data. PWVart with standard deviations >1 were excluded.
Nonfasted blood (Hb, HbA1c, plasma glucose, sodium, potassium, creatinine, aldosterone and renin mass concentrations) and urinary sodium, potassium, creatinine were measured by our accredited laboratory. Plasma nitrate and nitrite concentrations were measured by chemiluminescence as described.18, 25
2.2. Statistical analysis
Sufficient data from CAVI interventions were not available for sample size calculations. We used previous studies on BP with beetroot juice [33–34] and the 1‐year study of PWVart on spironolactone14 aiming to detect a 20% reduction over 6 months in PWV (standard deviation 8%) with minimum 80% power, at P < 0.05. We estimated we needed 24 participants per each of 4 arms, aiming for 30 per group allowing for 20% drop out, for 24 patients in each to finish the trial.
A modified intention‐to‐treat analysis was performed using SAS (version 9.3); data are presented as least‐square means estimated from mixed effects models (log‐transformed where not normally distributed), adjusted as prespecified for baseline, and any difference in final SBP change between the 2 arms being analysed. To estimate independence from BP change, changes in PWVart were adjusted for change in SBP. Least square mean data were averaged over the 2 follow‐up visits (3 and 6 months). Regression analyses assumed linear relationships, with some predictor variables (renin, nitrate and nitrite) log‐transformed.
3. RESULTS
3.1. Baseline
Of 154 patients agreeing to attend, 11 were not eligible (4 for high HbA1c, 2 for previous adverse reactions, 2 with atrial fibrillation, 3 for ill health) and 17 then declined to participate. The remaining 126 participants were randomised (Supplementary Figure 1). Baseline characteristics were generally well‐matched between arms (Table 1 and Supplementary Table 1) both between drugs and by nitrate/nitrate‐free juices (Tables 2, 3). Of randomised participants, 62% had T2DM with mean HbA1c 50 mM/M (6.7%). The remaining 38% were at risk (mean HbA1c <40 mM/M, 5.8%, body mass index 32.5 kg/m2).
Table 1.
Mean (with standard deviation) baseline characteristics of patients randomized into the VaSera trial
| Juice: | Spironolactone | Doxazosin | ||
|---|---|---|---|---|
| Active (n = 35) | Placebo (n = 30) | Active (n = 29) | Placebo (n = 33) | |
| Age (y) | 56.9 (13.9) | 56.3 (11.0) | 57.6 (12.3) | 55.9 (14.0) |
| Female, n (%) | 11 (31.4) | 11 (36.7) | 13 (44.8) | 11 (33.3) |
| Weight (kg) | 96.4 (15.0) | 94.8 (15.2) | 98.1 (19.7) | 88.4 (19.9) |
| Height (m) | 1.69 (0.10) | 1.70 (0.10) | 1.71 (0.10) | 1.72 (0.12) |
| Body mass index (kg/m 2 ) | 33.9 (4.9) | 33.0 (4.8) | 33.3 (6.1) | 30.2 (6.1) |
| Waist (cm) | 112 (29) | 110 (10) | 112 (13) | 103 (14) |
| T2DM, n (%) | 18 (51.4) | 20 (66.7) | 18 (62.1) | 22 (66.7) |
| eGFR (mL/min/1.73m 2 ) a | 81 (25) | 80 (17) | 84(23) | 84 (18) |
| Patients treated with | ||||
| Metformin, n (%) | 12 (36.4) | 15 (50.0) | 15 (51.7) | 15 (46.9) |
| Insulin, n (%) | 5 (14.7) | 8 (26.7) | 2 (6.9) | 8 (24.2) |
| Other oral diabetic medication, n (%) | 6 (17.6) | 7 (23.3) | 7 (24.1) | 6 (18.2) |
| Antihypertensive, n (%) b | 20 (57.1) | 20 (66.7) | 25 (86.2) | 24 (72.7) |
| Mean number of antihypertensives, n (%) c | 1.6 (0.7) | 1.9 (0.9) | 1.7 (0.8) | 1.7 (0.6) |
| Diuretics, n (%) | 5 (14.7) | 10 (33.3) | 9 (31.0) | 13 (40.6) |
| Statins, n (%) | 15 (44.1) | 15 (50.0) | 17 (58.6) | 15 (45.5) |
Active is nitrate‐containing beetroot juice; placebo is nitrate‐depleted beetroot juice.
Values are mean (standard deviation) unless stated otherwise.
Calculated using abbreviated MDRD equation,
represents those taking at least 1 antihypertensive,
represents mean number of antihypertensive drugs taken of those taking at least 1
eGFR, estimated glomerular filtration rate
Table 2.
Mean (with 95% confidence interval) vascular, plasma and urine parameters at baseline and follow‐up visits for spironolactone and doxazosin
| Baseline | Midpoint | End | ||||
|---|---|---|---|---|---|---|
| Spironolactone | Doxazosin | Spironolactone | Doxazosin | Spironolactone | Doxazosin | |
| Vascular | ||||||
| CAVI, units | 8.35 (8.02, 8.67) | 8.07 (7.68, 8.47) | 8.37 (8.08, 8.67) | 8.05 (7.57, 8.52) | 8.19 (7.81, 8.57) | 8.04 (7.56, 8.52) |
| PWVart, m/s | 9.4 (8.9, 9.9) | 9.7 (9.1, 10.2) | 9.3 (8.8, 9.7) | 8.9 (8.4, 9.4) | 9.3 (8.9, 9.7) | 9.3 (8.8, 9.9) |
| SBP, mmHg | 143.4 (138.3, 148.4) | 140.1 (136.2, 144.0) | 137.2 (132.4, 141.9) | 136.7 (132.1, 141.3) | 135.0 (131.1, 139.0) | 137.7 (132.7, 142.7) |
| DBP, mmHg | 88.0 (84.7, 91.2) | 86.8 (84.7, 88.9) | 84.5 (81.8, 87.1) | 84.5 (82.0, 87.1) | 82.6 (79.5, 85.7) | 84.8 (82.0, 87.7) |
| aoSBP, mmHg | 135.4 (129.1, 141.6) | 130.0 (123.7, 136.3) | 126.6 (120.5, 132.7) | 125.0 (119.5, 130.5) | 123.7 (118.3, 129.2) | 123.9 (119.2, 128.6) |
| brAIX, % | −16.4 (−24.8, ‐7.9) | −22.7 (−31.6, −13.8) | −22.4 (−30.2, −14.6) | −22.6 (−32.7, −12.4) | −24.6 (−32.9, −16.2) | −24.2 (−32.7, −15.7) |
| aoAIX, % | 29.4 (25.1, 33.6) | 26.1 (21.6, 30.6) | 26.3 (22.3, 30.2) | 26.2 (21.1, 31.4) | 25.2 (21.0, 29.4) | 25.4 (21.1, 29.7) |
| HR, beats/min | 69.9 (66.6, 73.2) | 71.7 (68.2, 75.2) | 69.3 (66.4, 72.1) | 69.6 (66.5, 72.6) | 71.4 (68.2, 74.5) | 69.6 (66.3, 72.9) |
| Plasma | ||||||
| Glucose,a mmol/L | 6.4 (5.8, 7.0) | 6.2 (5.6, 6.8) | 7.1 (6.3, 8.0) | 6.3 (5.6, 7.2) | 6.7 (5.8, 7.8) | 6.0 (5.3, 6.8) |
| HbA1c,a % | 6.7 (6.3, 7.0) | 6.7 (6.4, 7.0) | 6.9 (6.6, 7.3) | 6.7 (6.4, 7.1) | 6.8 (6.4, 7.2) | 6.7 (6.3, 7.1) |
| Sodium, mmol/L | 139.7 (139.0, 140.3) | 139.7 (139.0, 140.4) | 138.2 (137.4, 139.0) | 140.0 (139.3, 140.7) | 138.4 (137.5, 139.4) | 139.7 (139.0, 140.4) |
| Potassium, mmol/L | 4.27 (4.16, 4.39) | 4.18 (4.07, 4.30) | 4.53 (4.42, 4.64) | 4.21 (4.09, 4.34) | 4.60 (4.49, 4.72) | 4.17 (4.07, 4.28) |
| Creatinine a μmol/L | 81.6 (76.6, 87.0) | 81.4 (75.9, 87.3) | 83.1 (77.4, 89.3) | 83.7 (78.5, 89.2) | 84.7 (78.5, 91.4) | 83.4 (78.0, 89.1) |
| Renin,a mU/mL | 31.8 (18.7, 54.0) | 31.5 (20.2, 49.2) | 63.2 (38.5, 103.7) | 38.3 (21.6, 67.9) | 66.1 (39.3, 111.4) | 39.2 (22.1, 69.8) |
| Aldosterone,a pmol/L | 225 (191, 264) | 229 (199, 264) | 439 (367, 525) | 281 (241, 327) | 391 (325, 470) | 300 (251, 358) |
| Nitrate,a μM | 37.4 (29.6, 47.4) | 25.2 (19.0, 33.6) | 78.1 (55.8, 109.4) | 62.4 (43.5, 89.5) | 97.8 (66.5, 144.0) | 54.2 (35.9, 81.9) |
| Nitrite,a nM | 0.189 (0.123, 0.289) | 0.147 (0.098, 0.222) | 0.268 (0.177, 0.405) | 0.133 (0.076, 0.233) | 0.242 (0.146, 0.400) | 0.115 (0.065, 0.203) |
| Urine | ||||||
| Sodium,a mmol/L | 61.9 (53.1, 72.2) | 64.0 (52.7, 77.8) | 64.1 (53.8, 76.4) | 64.6 (53.1, 78.5) | 78.3 (65.5, 93.5) | 70.0 (58.8, 83.5) |
| Potassium,a mmol/L | 50.4 (43.4, 58.5) | 57.2 (48.3, 67.8) | 66.1 (56.5, 77.5) | 62.5 (53.3, 73.3) | 61.6 (52.0, 73.0) | 70.2 (60.8, 81.0) |
| Creatinine,a mmol/L | 7.99 (6.60, 9.68) | 9.21 (7.45, 11.38) | 8.86 (7.34, 10.70) | 10.57 (8.53, 13.08) | 8.09 (6.71, 9.75) | 10.58 (8.77, 12.76) |
Analysed in log units and geometric means presented.
PWVart, pulse wave velocity by Arteriograph; aoSBP, aortic blood pressure; aoAix aortic augmentation index; brAix, brachial augmentation index; CAVI, cardio−ankle vascular index; DBP, diastolic blood pressure; HR heart rate; SBP, systolic blood pressure. Values are mean (95% confidence interval)
Table 3.
Mean (with 95% confidence interval) vascular, plasma and urine parameters at baseline and 2 follow‐up visits for nitrate containing (active) and nitrate depleted (placebo) beetroot juice
| Baseline | Midpoint | End | ||||
|---|---|---|---|---|---|---|
| Active | Placebo | Active | Placebo | Active | Placebo | |
| Vascular | ||||||
| CAVI, units | 8.28 (7.92, 8.64) | 8.14 (7.77, 8.51) | 8.29 (7.91, 8.67) | 8.13 (7.72, 8.54) | 8.15 (7.71, 8.58) | 8.08 (7.65, 8.52) |
| PWVart, m/s | 9.7 (9.1, 10.3) | 9.4 (8.9, 9.8) | 9.3 (8.8, 9.8) | 8.9 (8.5, 9.4) | 9.5 (9.0, 9.9) | 9.2 (8.7, 9.7) |
| SBP, mmHg | 142.5 (137.7, 147.3) | 140.9 (136.7, 145.1) | 137.5 (132.8, 142.3) | 136.4 (131.7, 141.1) | 136.2 (131.9, 140.4) | 136.7 (131.9, 141.5) |
| DBP, mmHg | 88.5 (85.6, 91.5) | 86.3 (83.8, 88.7) | 84.4 (81.5, 87.2) | 84.6 (82.2, 87.0) | 83.4 (80.7, 86.1) | 84.1 (80.9, 87.3) |
| aoSBP, mmHg | 134.8 (128.8, 140.8) | 130.6 (124.0, 137.1) | 127.2 (121.1, 133.4) | 124.4 (118.9, 129.9) | 123.9 (119.2, 128.5) | 123.8 (118.3, 129.2) |
| brAIX, % | −13.5 (−21.5, −5.5) | −25.7 (−34.8, −16.6) | −20.4 (−28.7, −12.2) | −24.5 (−34.1, −14.8) | −23.2 (−31.4, −15.0) | −25.6 (−34.3, −16.9) |
| aoAIX, % | 30.8 (26.7, 34.9) | 24.6 (20.0, 29.2) | 27.3 (23.1, 31.5) | 25.3 (20.4, 30.1) | 25.9 (21.8, 30.0) | 24.7 (20.3, 29.1) |
| HR, beats/min | 68.5 (65.5, 71.5) | 73.0 (69.3, 76.7) | 68.8 (66.0, 71.7) | 69.9 (66.9, 73.0) | 71.8 (68.6, 75.1) | 69.1 (66.0, 72.2) |
| Plasma | ||||||
| Glucose a mmol/L | 5.91 (5.36, 6.52) | 6.64 (6.05, 7.29) | 7.04 (6.15, 8.05) | 6.45 (5.76, 7.21) | 6.49 (5.66, 7.45) | 6.21 (5.44, 7.08) |
| HbA1c,a % | 6.63 (6.30, 6.97) | 6.73 (6.43, 7.04) | 6.81 (6.42, 7.23) | 6.87 (6.57, 7.18) | 6.65 (6.24, 7.09) | 6.85 (6.52, 7.19) |
| Sodium, mmol/L | 139.9 (139.2, 140.5) | 139.5 (138.8, 140.2) | 138.8 (138.0, 139.7) | 139.3 (138.6, 140.1) | 139.1 (138.2, 140.0) | 139.0 (138.1, 139.8) |
| Potassium, mmol/L | 4.26 (4.15, 4.37) | 4.20 (4.08, 4.33) | 4.46 (4.32, 4.59) | 4.32 (4.20, 4.43) | 4.41 (4.28, 4.55) | 4.38 (4.26, 4.49) |
| Creatinine a μmol/L | 81.3 (75.4, 87.6) | 81.7 (77.1, 86.7) | 83.4 (77.1, 90.3) | 83.4 (78.8, 88.2) | 85.3 (78.6, 92.5) | 82.9 (78.1, 87.9) |
| Renin,a mU/mL | 23.5 (14.7, 37.6) | 42.6 (26.0, 69.8) | 41.6 (24.7, 70.0) | 57.4 (33.0, 99.7) | 49.8 (29.6, 83.7) | 54.7 (30.4, 98.6) |
| Aldosterone,a pmol/L | 230 (200, 266) | 224 (190, 263) | 337 (281, 406) | 363 (305, 431) | 375 (318, 442) | 319 (260, 391) |
| Nitrate,a μM | 28.8 (22.5, 36.9) | 32.4 (24.2, 43.4) | 125.4 (94.0, 167.3) | 43.4 (32.5, 58.1) | 118.0 (82.4, 169.0) | 35.9 (25.7, 50.0) |
| Nitrite,a nM | 0.191 (0.130, 0.282) | 0.144 (0.092, 0.226) | 0.268 (0.168, 0.428) | 0.139 (0.083, 0.233) | 0.219 (0.135, 0.353) | 0.107 (0.056, 0.203) |
| Urine | ||||||
| Sodium,a mmol/L | 66.2 (54.4, 80.6) | 60.0 (51.7, 69.6) | 66.9 (54.9, 81.5) | 61.8 (52.0, 73.3) | 79.7 (67.3, 94.3) | 68.9 (57.3, 82.9) |
| Potassium,a mmol/L | 51.6 (44.2, 60.1) | 55.4 (47.0, 65.3) | 63.0 (53.2, 74.7) | 65.3 (56.3, 75.8) | 65.8 (56.9, 76.0) | 65.6 (55.1, 78.1) |
| Creatinine,a mmol/L | 8.96 (7.35, 10.93) | 8.18 (6.67, 10.03) | 9.58 (7.82, 11.73) | 9.77 (7.97, 11.98) | 9.28 (7.68, 11.22) | 9.12 (7.51, 11.06) |
Analysed in log units and geometric means presented
Active is nitrate containing beetroot juice; placebo is nitrate depleted beetroot juice. PWVart, pulse wave velocity by Arteriograph; aoSBP, aortic blood pressure; aoAix aortic augmentation index; brAix, brachial augmentation index; CAVI, cardio−ankle vascular index; DBP, diastolic blood pressure; HR heart rate; SBP, systolic blood pressure. Values are mean (95% confidence interval)
3.2. Follow up
Time from randomization to midpoint dose increase was 13 ± 3 weeks and from midpoint‐final visit 12 ± 3 weeks, totalling 24 ± 5 weeks from randomisation to end‐of‐study. Between baseline and 12 weeks' follow‐up, 16 participants dropped out (6 no reason, 1 unrelated illness, 4 not re‐contacted and 5 with side effects: 2 dizziness, 2 elevated glucose, 1 breathlessness). There were no follow‐up measures for these participants.
3.3. Treatment effects
No statistical interactions occurred between beetroot or placebo juice arms and the spironolactone vs doxazosin arm for any of the main/haemodynamic outcomes, so data are presented separately (Tables 2, 3). Supplementary Table 1 shows absolute, unadjusted changes of vascular and biological parameters for the 4 arms.
Spironolactone vs doxazosin: In adjusted models, spironolactone and doxazosin reduced BP similarly (SBP, least‐square mean [95% CI]: −7.0 [−9.9, −4.2] vs ‐6.3 [−9.1, −3.5] mmHg respectively, P = .7; Figure 1C and diastolic BP, −5.6 [−7.4, −3.7] vs ‐4.7 [−6.5, −2.9] mmHg respectively, P = .5; Supplementary Figure 2A). The direction in difference for the primary endpoint, change in CAVI between drugs, was contrary to our hypothesis, borderline significant towards doxazosin (0.14 [−0.06, 0.34] vs –0.11 [−0.30, 0.08] units P = .08, for spironolactone and doxazosin, respectively; Figure 1A). When transposed to CAVI0, our data was not significant −0.04(−0.44, 0.35) vs 0.24 (−0.19, 0.67), P = .34 (doxazosin vs spironolactone).19 However, the difference in PWVart change between spironolactone and doxazosin was significant (−0.07 [−0.33, 0.18] vs −0.44 [−0.69, −0.19] m/s, P = .045, Figure 1B) towards doxazosin, again contrary to our hypothesis. There were also no other differences in other haemodynamic parameters estimated by the Arteriograph for the drug arm, in central BP (−7.6 [−9.0, −6.3] vs ‐7.2 [−8.5, −5.9] mmHg, P = .6; Figure 1 D), augmentation index (Supplementary Figure 2 B‐C), or heart rate.
Figure 1.
Change in vascular parameters in response to spironolactone and doxasozin. Change in cardio−ankle vascular index, aortic pulse wave velocity, systolic, and central blood pressure and plasma nitrate and nitrite concentration on drug intervention. Data are least square means averaged over the 2 follow‐up visits with mean, 95% confidence intervals. *P < .05. (A) cardio−ankle vascular index (CAVI); (B) pulse wave velocity by Arteriograph (PWV); (C) systolic blood pressure (SBP); (D) aortic SBP (aoSBP); E plasma nitrate concentration [Nitrate]; (F) plasma nitrite concentration [Nitrite]
Although no drug/juice interactions in terms of haemodynamic variables were noted, nitrate and nitrite concentrations were higher on spironolactone than on doxazosin by 1.5‐fold [1.1, 2.2] and 2.2‐fold [1.3, 3.6] respectively; both P < .05; see Figure 1 E‐F. Unadjusted data are in Supplementary Table 1.
Beetroot vs placebo juice: There were no adjusted differences in change in arterial stiffness change as CAVI (0.02 [−0.18, 0.21] vs 0.01 [−0.18, 0.21], P = .98; Figure 2A) CAVI0 0.12(−0.29, 0.53) vs 0.08(−0.34, 0.50), P = .898 (active vs control)19 nor PWVart (−0.23 [−0.48, 0.01] vs −0.28 [−0.54, −0.03], P = .8; Figure 2B), nor in brachial BP between active and placebo juice (SBP, −6.4 [−9.2, −3.6] vs −6.9 [−9.8, −4.0] mmHg, P = .8, Figure 2C, nor diastolic BP, P = .9 (Supplementary Figure 3A). However, difference in change in central (aortic) SBP between active and control juices was highly significant (−8.7 [−10, −7.4] vs −6.1 [−7.4, −4.8] mmHg, P = .007; Figure 2D). Decreases in aortic (−3 [−5.1, −0.9] vs −0.3 [−2.4, 1.9] %, P = .08) and brachial augmentation index (−5.9 [−10.0, −1.76] vs −0.49 [−4.72, 3.74] %, P = .08) were also borderline (Supplementary Figure 3B, C).
Figure 2.
Change in vascular parameters in response to inorganic nitrate from beetroot juice and nitrate free, placebo beetroot juice. Change in cardio–ankle vascular index, aortic pulse wave velocity, systolic, and central blood pressure, aortic and brachial augmentation index and plasma nitrate and nitrite concentration on juice intervention. Data are least square means averaged over the 2 follow‐up visits with mean, 95% confidence intervals. *P > .05, **P < .01, ***P < .001. (A) cardio−ankle vascular index (CAVI); (B) pulse wave velocity by Arteriograph (PWV); (C) systolic blood pressure (SBP); (D) aortic SBP (aoSBP); E plasma nitrate concentration [Nitrate]; (F) plasma nitrite concentration [Nitrite]
Plasma nitrate levels rose as expected in those on active compared with placebo juice (a 4.3 [3.4, 5.5]‐fold increase vs 1.3 [1.02, 1.71], P < .001, Figure 2 E); nitrite levels increased 1.6 [1.1, 2.2]‐fold vs 0.9 [0.6, 1.2], P = .02, Figure 2 F). These data confirm adherence to the beetroot juice arm. Unadjusted data are in Supplementary Table 1.
3.4. Adverse effects
From randomization, all adverse effects were documented and assessed after unblinding, which did not occur until after the trial finished. Of 126 participants randomized, 12 reported effects deemed to be related to the drug interventions (5 dizziness of whom 4 taking doxazosin, 2 rashes [1 taking spironolactone], 1 reported incontinence [doxazosin], nausea [spironolactone], heartburn [spironolactone], tachycardia [doxazosin], breathlessness [spironolactone]). In 8 of these patients, doses were adjusted or stopped; 1 participant willingly tolerated the effects. Throughout the study, 5 patients withdrew consent due to adverse effects, 3 deemed related to the intervention (2 dizziness, 1 breathlessness); 1 patient reported dyspepsia, deemed related to juice. No participants were excluded.
3.5. Further regression analyses
Relationships between baseline plasma renin, nitrate and nitrite and change in CAVI, PWVart and central BP (from baseline to follow‐up) were examined. Change in central BP was significantly related to baseline plasma renin (r = .36, P < .001), so that for a 10‐fold reduction in plasma renin, there was an 8.6 mmHg greater decrease in central BP (Figure 3C). This result was not specific to either the drug or juice arm. There were no relationships between change in CAVI or PWVart and renin (r = .02, P = .8, and r = .05, P = .6, respectively; Figure 3A‐B). There were also no relationships between change in CAVI, PWVart or central BP and nitrate (Supplementary Figure 4 A–C; r = .07, P = .6; r = −.04, P = .7; r = .01, P = .9, respectively) or nitrite (Supplementary Figure 5A‐C; r = .13, P = .3; r = .02, P = .87; r = −.06, P = 0.7, respectively).
Figure 3.
Correlation between change in vascular parameters and baseline plasma renin. Change in: (A) cardio–ankle vascular index (CAVI); (B) pulse wave velocity by Arteriograph (PWV); and (C) aortic systolic blood pressure (aoSBP) vs baseline plasma nitrite concentration, n = 64
4. DISCUSSION
This randomised trial demonstrated a proof of concept that reduction of arterial stiffness, an independent predictor of mortality generally and in T2DM,4 could be measured and estimated independently of BP, as measured by CAVI and PWVart, adjusting for differences in achieved BP between trial groups.
4.1. Spironolactone vs doxazosin
The reduction in CAVI, which measures cardiac–ankle PWV, including a long more muscular arterial path, was borderline (P = .07). However, contrary to our hypothesis, the result was in the opposite direction, towards the doxazosin, not the spironolactone arm. The consistency and direction of this effect on arterial stiffness was supported, again independent of BP change, by the significant impact on central PWVart, our other main outcome. Unlike aortic PWV measured as carotid–femoral3 or down just the descending aorta,6 PWV in the extremities, down muscular arterial pathways such as the femoral–posterior tibial or cardiac–brachial routes, does not predict outcomes.33 However, the ease of CAVI/PWV measurement using the multicuff arm–ankle method, which includes the central aorta, and the microphone‐detected second sound timing for the precise initiation of the pressure/flow wave outweighs issues of including extremity pathways in its measurements. BP independence of CAVI has been discussed and re‐formulated to produce CAVI0 32, 34; when we transposed our data based on CAVI0 suggested by Spronk et al., they were not significant.35
Here, adequate daily doses of ≤16 mg doxazosin, as α‐receptor blockade, were compared with ≤50 mg spironolactone, as mineralocorticoid receptor antagonist. Our results contrast with previous work, which suggested spironolactone at just 25 mg reduced PWV by 0.8 m/s vs placebo, apparently with little change in bp,14 in patients with mild kidney impairment; in that study, spironolactone had been added to angiotensin converting enzyme inhibitors and angiotensin receptor blockers. While the change in PWV and aortic distensibility was significant, so also was the change in either 24h ambulatory, or in office systolic BP; i.e. 1 was not independent of the other. Left ventricular mass also changed, probably in response to the decreased BP. In our study, we found that left ventricular mass index between the 2 active BP drugs was not significant.36 Here, 71% patients were on prior anti‐hypertensive medication of many types, and the 62% with T2DM generally on metformin and other glucocentric agents. The difference in change of (office) BP was not significant, despite adjusting for the small change in favour of spironolactone. Although there are suggestions that doxazosin may reduce arterial stiffness,37, 38 neither of those studies was a formal trial nor adjusted for any BP change, and its use in arterial function has not to our knowledge been examined in T2DM. From a physiological point of view the action of doxazosin can be easily explained. Vascular tone does influence arterial stiffness in muscular arteries39, 40 and is likely to have a similar action in larger arteries (although this influence is difficult to assess due to concomitant effect in BP).
The absolute reduction in BP for those who finished 6 months' treatment was a similar 7 mmHg SBP reduction in both drug groups, but the least square mean fall in BP was a nonsignificantly 2.3 mmHg greater on spironolactone than doxazosin, using a higher dose than in our recent blinded, rotational pathway trial where the difference was 4.5 mmHg.13 The different patient population and the lower dose of doxazosin probably contributed to different treatment responses there to here.
Results from the antihypertensive ALLHAT Trial are relevant; its doxazosin arm had to be stopped after ~2 years, due to excess heart failure and other cardiac events.41 Having diabetes on doxazosin in the trial was a particular aggravating factor.42 Whilst the change in PWVart and the borderline change in CAVI could be related to changes in cardiac function, our echocardiographic data36 do not suggest that as the ejection fraction, and global longitudinal strain, which is a well‐established marker of systolic function, were similar between the 2 drugs in our study; however, S′ (a tissue‐Doppler systolic function index) was increased by spironolactone vs doxazosin. Thus, while our data suggest that we have shown proof of concept that PWVart can be reduced independent of BP change, we have not shown that it is independent of cardiac functional change.
4.2. Effects of inorganic nitrate
No effect of active (nitrate containing) beetroot juice, even at higher dose, was found on peripheral (brachial) BP, CAVI or PWV consistent with 2 previous dietary nitrate studies in patients with diabetes21, 22 and in line with our recent results that acute physiological elevations of plasma glucose and insulin, following an oral glucose tolerance test, result in a lack of BP‐lowering with dietary nitrate in healthy adults.43 Previous reductions in PWV were with peripheral BP reductions22; the lack of change in peripheral BP with nitrate may underlie the lack of effect on PWV, suggesting dietary nitrate has no direct effect on arterial stiffness. Further the lack of reduction on PWV with dietary nitrate is in line with acute effects seen previously with glyceryl trinitrate.44 Plasma nitrate and nitrite did increase, some 4‐fold and only 2‐fold respectively. The 2 other diabetes studies21, 22 also found significant increases in plasma nitrite similar to that in healthy participants18 and hypertensives.19
Central SBP decreased on nitrate‐containing juice, with similar if borderline changes in augmentation index, simultaneous to the significant rise in plasma nitrite, without peripheral BP changes. Although this could be as a result of venodilation with reduced preload, indeed decreased central SBP was observed with decreased preload (induced by lower limb venous occlusion),45 in an echocardiogram substudy (data not presented here), we saw only very small differences in stroke volume between treatments and so, although it might be contributory this is unlikely to be an alternative mechanism.36 This selective central SBP change is entirely consistent with our previous findings of normoxia‐dependent conduit artery dilatation after inorganic nitrite, selectively reducing central SBP.28 A measurable increase in plasma nitrite in healthy volunteers also led to decreased brachial‐femoral PWV, independently of peripheral BP.28 A different more muscular brachial conduit artery arterial path was studied there. However, whether currently measured central BP has clinical impact beyond peripheral BP in the general population, as some claim,46 remains uncertain, as recently reported from Framingham,47 in part related to calibration issues.48, 49 However, central aortic pressure may be especially relevant in specific populations, such as those with heart failure with preserved ejection fraction.50
The confirmation of a central BP effect here, as found previously, suggests that testing for central aortic stiffening changes, affecting the aortic root, ascending aorta or arch using other imaging methods including magnetic resonance imaging could be revealing. Other recent Framingham work confirms that rather than flow‐mediated dilation per se, poorer forearm hyperaemic mean blood flow velocity reflecting microvascular (smaller resistance vessel) changes underlies some 8–13% of the overall stiffening effect measured by PWV that predicts outcomes powerfully and independently of BP in that cohort.51
Despite observing no drug/juice interactions in haemodynamic parameters, there was an interesting finding of increased plasma nitrate and nitrite concentrations observed on spironolactone vs doxazosin. This could be related to spironolactone's diuretic effect, haemoconcentrating nitrate and nitrite, relative to the vasodilatory effect of doxazosin, or via altering renal nitrate/nitrite excretion; unfortunately, the latter was not assessed in this study.
Adverse events attributable to the blinded interventions were small and minor, with 1 person mentioning some increased reflux/acidity on the active, nitrate containing beetroot juice. Potassium retention on spironolactone was not a problem at all, probably because entry excluded people with estimated glomerular filtration rates of ≤45 mL/min.
This was intentionally a pragmatic trial testing general efficacy of the interventions. In retrospect, the choice of doxazosin as the control antihypertensive agent for spironolactone could be disputed, but few other drugs currently balance dosage and effect equivalently. Medication timing over the 6 months, and adherence to respective treatments could not be assured, although changes in nitrate concentrations on active juice suggested reasonable adherence to the juice overall. Participants were asked to take their treatment and juice on rising or around breakfast time, since peak plasma nitrite concentrations after dietary nitrate ingestion occurs about 2.5 h later18; however, the intervals between juice ingestion and visits to the Clinical Research Facility and hence blood collection may have been highly variable. Measurements were all made under standardised conditions in a Clinical Research Facility. We also recognize the limitation in our sample size calculation being based on PWV, and not CAVI, the primary outcome of the research; this was due to sufficient data not being available at the time of starting the trial.
Contrary to our hypothesis, arterial stiffness was not reduced on spironolactone, rather that occurred on the doxazosin arm independently of BP, as measured by PWVart, with a similar borderline effect on the longer muscular arterial pathway estimated by CAVI, in these patients with or at risk of T2DM. Whilst active nitrate‐containing beetroot juice had no effect on arterial stiffness, central BP was significantly reduced by nitrate–nitrite.
COMPETING INTERESTS
A.J.W. holds shares in HeartBeet Ltd, which receive a royalty from James White Drinks Ltd who manufacture the active nitrate‐containing beetroot juice and placebo nitrate‐depleted juice used in this study. The other authors have stated explicitly that there are no conflicts of interest in connection with this article. This work was funded by Fukuda Denshi, Tokyo, Japan.
CONTRIBUTORS
A.J.W. and J.K.C both led the design of the research and oversaw the acquisition of the data and data analysis, they were involved in the interpretation of the results and revising the manuscript drafts. C.E.M. contributed to research design, she led the data acquisition and was involved in the interpretation of the results and led drafting the manuscript. V.G., L.F. and M.L.C. were all involved in research design, data acquisition and interpretation and revising the manuscript drafts. S.V.M. lead the data analysis and contributed to the manuscript drafts. H.C., F.I., P.M., A.M. and E.N. were all involved in data acquisition and contributed to manuscript drafts. All authors approved the final version of the manuscript and have agreed accountability of the research.
Supporting information
TABLE S1 Mean (with 95% confidence interval) vascular, plasma and urine parameters at baseline and 2 follow‐up visits for combination of spironolactone or doxazosin and nitrate containing (active) or nitrate depleted (placebo) beetroot juice
FIGURE S1 CONSORT* flow diagram for VaSera trial. Flow diagram detailing the recruitment and retention in study as well as number of measures analysed
FIGURE S2 Change in vascular parameters in response to spironolactone and doxasozin
FIGURE S3 Change in vascular parameters in response to inorganic nitrate from beetroot juice and nitrate free, placebo beetroot juice
FIGURE S4 Correlation between change in vascular parameters and baseline plasma nitrate
FIGURE S5 Correlation between change in vascular parameters and baseline plasma nitrite
ACKNOWLEDGEMENTS
The authors thank the research nurses at Clinical Research Facility at St Thomas' Hospital for their assistance in running the study and the patients who participated in the study. We also thank Karen McNeill for training and supervising the chemiluminescence analysis of the plasma and juices and for managing the blinding and randomization of the interventions. Finally, we thank Suzanne Barrett who worked as research administrator for all her help in ensuring the smooth running of the trial.
We acknowledge internal infrastructure financial support from King's College London British Heart Foundation Centre; National Institute for Health Research (NIHR), Clinical Research Facility at Guy's & St Thomas' NHS Foundation Trust and NIHR Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Lastly, we give thanks to Fukuda Denshi who funded this work.
Mills CE, Govoni V, Faconti L, et al. A randomised, factorial trial to reduce arterial stiffness independently of blood pressure: Proof of concept? The VaSera trial testing dietary nitrate and spironolactone. Br J Clin Pharmacol. 2020;86:891–902. 10.1111/bcp.14194
PI statement: The authors confirm that the Principal Investigator for this paper is Professor J.K. Cruickshank and that he had direct clinical responsibility for patients.
Clinical trial registration: ISRCTN registry: ISRCTN25003627/DOI https://doi.org/10.1186/ISRCTN25003627.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
REFERENCES
- 1. Selvin E, Steffes MW, Zhu H, et al. Glycated hemoglobin, diabetes, and cardiovascular risk in nondiabetic adults. N Engl J Med. 2010;362(9):800‐811. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Retnakaran R, Shah BR. Role of type 2 diabetes in determining retinal, renal, and cardiovascular outcomes in women with previous gestational diabetes mellitus. Diabetes Care. 2017;40(1):101‐108. [DOI] [PubMed] [Google Scholar]
- 3. Ben‐Shlomo Y, Spears M, Boustred C, et al. Aortic pulse wave velocity improves cardiovascular event prediction: an individual participant meta‐analysis of prospective observational data from 17,635 subjects. JACC. 2014;63(7):636‐646. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Cruickshank K, Riste L, Anderson SG, Wright JS, Dunn G, Gosling RG. Aortic pulse‐wave velocity and its relationship to mortality in diabetes and glucose intolerance: an integrated index of vascular function? Circulation. 2002;106(16):2085‐2090. [DOI] [PubMed] [Google Scholar]
- 5. Eliakim M, Sapoznikov D, Weinman J. Pulse wave velocity in healthy subjects and in patients with various disease states. Am Heart J. 1971;82(4):448‐457. [DOI] [PubMed] [Google Scholar]
- 6. Gunn GC, Dobson HL, Gray J, Geddes LA, Vallbona C. Studies of pulse wave velocity in potential diabetic subjects. Diabetes. 1965;14:489‐492. [DOI] [PubMed] [Google Scholar]
- 7. Bramwell JC, Hill AV. Velocity of transmission of the pulse‐wave and elasticity of arteries. Lancet. 1922;199(5149):891‐892. [Google Scholar]
- 8. Prenner SB, Chirinos JA. Arterial stiffness in diabetes mellitus. Atherosclerosis. 2015;238(2):370‐379. [DOI] [PubMed] [Google Scholar]
- 9. Freel EM, Mark PB, Weir RA, et al. Demonstration of blood pressure‐independent noninfarct myocardial fibrosis in primary aldosteronism: a cardiac magnetic resonance imaging study. Circ Cardiovasc Imaging. 2012;5(6):740‐747. [DOI] [PubMed] [Google Scholar]
- 10. Raaz U, Schellinger IN, Chernogubova E, et al. Transcription factor RUNX2 promotes aortic fibrosis and stiffness in type 2 diabetes mellitus. Circ Res. 2015;117(6):513‐524. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Hung CS, Chou CH, Liao CW, et al. Aldosterone induces tissue inhibitor of metalloproteinases‐1 expression and further contributes to collagen accumulation: from clinical to bench studies. Hypertension. 2016;67(6):1309‐1320. [DOI] [PubMed] [Google Scholar]
- 12. Laurent S, Cockcroft J, Van Bortel L, et al. Expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur Heart J. 2006;27(21):2588‐2605. [DOI] [PubMed] [Google Scholar]
- 13. Williams B, MacDonald TM, Morant S, et al. Spironolactone versus placebo, bisoprolol, and doxazosin to determine the optimal treatment for drug‐resistant hypertension (PATHWAY‐2): a randomised, double‐blind, crossover trial. Lancet. 2015;386(10008):2059‐2068. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Edwards NC, Steeds RP, Stewart PM, Ferro CJ, Townend JN. Effect of spironolactone on left ventricular mass and aortic stiffness in early‐stage chronic kidney disease: a randomized controlled trial. JACC. 2009;54(6):505‐512. [DOI] [PubMed] [Google Scholar]
- 15. Mahmud A, Feely J. Aldosterone‐to‐renin ratio, arterial stiffness, and the response to aldosterone antagonism in essential hypertension. Am J Hypertens. 2005;18(1):50‐55. [DOI] [PubMed] [Google Scholar]
- 16. Vizzardi E, Pina PD, Caretta G, et al. The effect of aldosterone‐antagonist therapy on aortic elastic properties in patients with nonischemic dilated cardiomyopathy. J Cardiovasc Med. 2015;16(9):597‐602. [DOI] [PubMed] [Google Scholar]
- 17. Lidder S, Webb AJ. Vascular effects of dietary nitrate (as found in green leafy vegetables and beetroot) via the nitrate‐nitrite‐nitric oxide pathway. Br J Clin Pharmacol. 2013;75(3):677‐696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Webb AJ, Patel N, Loukogeorgakis S, et al. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension. 2008;51(3):784‐790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Kapil V, Khambata RS, Robertson A, Caulfield MJ, Ahluwalia A. Dietary nitrate provides sustained blood pressure lowering in hypertensive patients: a randomized, phase 2, double‐blind, placebo‐controlled study. Hypertension. 2015;65(2):320‐327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Khatri J, Mills CE, Maskell P, Odongerel C, Webb AJ. It is rocket science ‐ why dietary nitrate is hard to 'beet'! Part I: twists and turns in the realization of the nitrate‐nitrite‐NO pathway. Br J Clin Pharmacol. 2017;83(1):129‐139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Shepherd AI, Gilchrist M, Winyard PG, et al. Effects of dietary nitrate supplementation on the oxygen cost of exercise and walking performance in individuals with type 2 diabetes: a randomized, double‐blind, placebo‐controlled crossover trial. Free Radic Biol Med. 2015;86:200‐208. [DOI] [PubMed] [Google Scholar]
- 22. Gilchrist M, Winyard PG, Aizawa K, Anning C, Shore A, Benjamin N. Effect of dietary nitrate on blood pressure, endothelial function, and insulin sensitivity in type 2 diabetes. Free Radic Biol Med. 2013;60:89‐97. [DOI] [PubMed] [Google Scholar]
- 23. Ashor AW, Lara J, Siervo M. Medium‐term effects of dietary nitrate supplementation on systolic and diastolic blood pressure in adults: a systematic review and meta‐analysis. J Hypertens. 2017;35(7):1353‐1359. [DOI] [PubMed] [Google Scholar]
- 24. Ghosh SM, Kapil V, Fuentes‐Calvo I, et al. Enhanced vasodilator activity of nitrite in hypertension: critical role for erythrocytic xanthine oxidoreductase and translational potential. Hypertension. 2013;61(5):1091‐1102. [DOI] [PubMed] [Google Scholar]
- 25. Omar SA, Fok H, Tilgner KD, et al. Paradoxical normoxia‐dependent selective actions of inorganic nitrite in human muscular conduit arteries and related selective actions on central blood pressures. Circulation. 2015;131(4):381‐389. [DOI] [PubMed] [Google Scholar]
- 26. Mills CE, Khatri J, Maskell P, Odongerel C, Webb AJ. It is rocket science ‐ why dietary nitrate is hard to 'beet'! Part II: further mechanisms and therapeutic potential of the nitrate‐nitrite‐NO pathway. Br J Clin Pharmacol. 2017;83(1):140‐151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. O'Gallagher K, Khan F, Omar SA, et al. Inorganic nitrite selectively dilates epicardial coronary arteries. JACC. 2018;71(3):363‐364. [DOI] [PubMed] [Google Scholar]
- 28. Omar SA, Artime E, Webb AJ. A comparison of organic and inorganic nitrates/nitrites. Nitric Oxide. 2012;26(4):229‐240. [DOI] [PubMed] [Google Scholar]
- 29. Webb AJ, Ahluwalia A. Mechanisms of nitrite reduction in ischemia in the cardiovascular system: therapeutic potential. Second ed. London: Academic Press; 2010. [Google Scholar]
- 30. Mills CE, Govoni V, Casagrande ML, Faconti L, Webb AJ, Cruickshank JK. Design and progress of a factorial trial testing the effect of spironolactone and inorganic nitrate on arterial function in people at risk of or with type 2 diabetes. Artery Res. 12:48‐53. [Google Scholar]
- 31. Shirai K, Utino J, Otsuka K, Takata M. A novel blood pressure‐independent arterial wall stiffness parameter; cardio‐ankle vascular index (CAVI). J Atheroscler Thromb. 2006;13(2):101‐107. [DOI] [PubMed] [Google Scholar]
- 32. Spronck B, Avolio AP, Tan I, Butlin M, Reesink KD, Delhaas T. Arterial stiffness index beta and cardio‐ankle vascular index inherently depend on blood pressure but can be readily corrected. J Hypertens. 2017;35(1):98‐104. [DOI] [PubMed] [Google Scholar]
- 33. Kapil V, Milsom AB, Okorie M, et al. Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite‐derived NO. Hypertension. 2010;56(2):274‐281. [DOI] [PubMed] [Google Scholar]
- 34. Mestanik M, Jurko A, Spronck B, et al. Improved assessment of arterial stiffness using corrected cardio‐ankle vascular index (CAVI0) in overweight adolescents with white‐coat and essential hypertension. Scand J Clin Lab Invest. 2017;77(8):665‐672. [DOI] [PubMed] [Google Scholar]
- 35. Mills CE, Govoni V, Faconti L, et al. Reducing arterial stiffness independently of blood pressure: the VaSera trial. JACC. 2017;70(13):1683‐1684. [DOI] [PubMed] [Google Scholar]
- 36. Faconti L, Mills CE, Govoni V, et al. Cardiac effects of 6 months' dietary nitrate and spironolactone in patients with hypertension and with/at risk of type 2 diabetes, in the factorial design, double‐blind, randomized controlled VaSera trial. Br J Clin Pharmacol. 2019;85(1):169‐180. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37. Komai N, Ohishi M, Moriguchi A, et al. Low‐dose doxazosin improved aortic stiffness and endothelial dysfunction as measured by noninvasive evaluation. Hypertens Res. 2002;25(1):5‐10. [DOI] [PubMed] [Google Scholar]
- 38. Wykretowicz A, Guzik P, Krauze T, Adamska K, Milewska A, Wysocki H. Add‐on therapy with doxazosin in patients with hypertension influences arterial stiffness and albuterol‐mediated arterial vasodilation. Br J Clin Pharmacol. 2007;64(6):792‐795. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39. Dobrin PB, Rovick AA. Influence of vascular smooth muscle on contractile mechanics and elasticity of arteries. Am J Physiol. 1969;217(6):1644‐1651. [DOI] [PubMed] [Google Scholar]
- 40. Boutouyrie P, Lacolley P, Girerd X, Beck L, Safar M, Laurent S. Sympathetic activation decreases medium‐sized arterial compliance in humans. Am J Physiol. 1994;267(4 Pt 2):H1368‐H1376. [DOI] [PubMed] [Google Scholar]
- 41. Davis BR, Kostis JB, Simpson LM, et al. Heart failure with preserved and reduced left ventricular ejection fraction in the antihypertensive and lipid‐lowering treatment to prevent heart attack trial. Circulation. 2008;118(22):2259‐2267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42. Barzilay JI, Davis BR, Bettencourt J, et al. Cardiovascular outcomes using doxazosin vs. chlorthalidone for the treatment of hypertension in older adults with and without glucose disorders: a report from the ALLHAT study. J Clinc Hypertens. 2004;6(3):116‐125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43. Floyd CN, Lidder S, Hunt J, Omar SA, McNeill K, Webb AJ. Acute interaction between oral glucose (75 g as Lucozade) and inorganic nitrate: decreased insulin clearance, but lack of blood pressure‐lowering. Br J Clin Pharmacol. 2019;85(7):1443‐1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44. Yaginuma T, Avolio A, O'Rourke M, et al. Effect of glyceryl trinitrate on peripheral arteries alters left ventricular hydraulic load in man. Cardiovasc Res. 1986;20:153‐160. [DOI] [PubMed] [Google Scholar]
- 45. Faconti L, Farukh B, McNally R, Webb A, Chowienczyk P. Arterial stiffness can be modulated by pressure‐independent mechanisms in hypertension. J Am Heart Assoc. 2019;8(15):e012601. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46. McEniery CM, Cockcroft JR, Roman MJ, Franklin SS, Wilkinson IB. Central blood pressure: current evidence and clinical importance. Eur Heart J. 2014;35(26):1719‐1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47. Mitchell GF, Hwang SJ, Larson MG, et al. Transfer function‐derived central pressure and cardiovascular disease events: the Framingham heart study. J Hypertens. 2016;34(8):1528‐1534. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48. Sharman JE. Central pressure should be used in clinical practice. Artery Res. 9:1‐7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49. Sharman JE, Avolio AP, Baulmann J, et al. Validation of non‐invasive central blood pressure devices: ARTERY society task force consensus statement on protocol standardization. Eur Heart J. 2017;38(37):2805‐2812. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50. Zamani P, Rawat D, Shiva‐Kumar P, et al. Effect of inorganic nitrate on exercise capacity in heart failure with preserved ejection fraction. Circulation. 2015;131(4):371‐380. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51. Cooper LL, Palmisano JN, Benjamin EJ, et al. Microvascular function contributes to the relation between aortic stiffness and cardiovascular events: the Framingham heart study. Circ Cardiovasc Imaging. 2016;9(12):e004979. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
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Supplementary Materials
TABLE S1 Mean (with 95% confidence interval) vascular, plasma and urine parameters at baseline and 2 follow‐up visits for combination of spironolactone or doxazosin and nitrate containing (active) or nitrate depleted (placebo) beetroot juice
FIGURE S1 CONSORT* flow diagram for VaSera trial. Flow diagram detailing the recruitment and retention in study as well as number of measures analysed
FIGURE S2 Change in vascular parameters in response to spironolactone and doxasozin
FIGURE S3 Change in vascular parameters in response to inorganic nitrate from beetroot juice and nitrate free, placebo beetroot juice
FIGURE S4 Correlation between change in vascular parameters and baseline plasma nitrate
FIGURE S5 Correlation between change in vascular parameters and baseline plasma nitrite
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.