1. Introduction
Cardiovascular diseases are the leading cause of morbidity and mortality worldwide [
1] and there is considerable interest in strategies to promote healthy aging. Several factors contribute to the age-related decline in cardiovascular health, and a central determinant is the bioavailability of nitric oxide (NO). Nitric oxide is the primary regulator of vascular tone and it has an essential role in the prevention of platelet aggregation, inhibition of vascular smooth muscle cell proliferation, and the prevention of atherosclerotic plaque formation [
2,
3,
4]. Older adults demonstrate reduced NO bioavailability [
5], which contributes to age-related increases in blood pressure and vascular stiffness [
6] and the associated risk of cardiovascular events [
3,
7]. Impairments in NO bioavailability may also lead to a reduction in limb blood flow, possibly through the role of NO in functional sympatholysis [
8] and endothelium-dependent vasodilation [
9,
10]. The decrease in blood flow is particularly evident in the legs [
11,
12] and is associated with reduced muscle function and diminished exercise capacity. Age-related reductions in leg blood flow are exacerbated in those with chronic conditions such as peripheral arterial disease [
13] and heart failure [
14]. Strategies to improve NO bioavailability, and thereby improve vascular function and enhance leg blood flow, may help in the prevention and management of age-related cardiovascular impairments.
Vascular NO is produced endogenously from L-arginine via endothelial nitric oxide synthase (eNOS), but it can also be generated from exogenous supplementation via the diet. Natural food sources high in inorganic nitrate (NO
3−), such as beetroot (
Beta vulgaris), can be reduced to nitrite (NO
2−) by oral bacteria [
15,
16,
17] and further reduced to NO by a wide variety of enzymatic and non-enzymatic pathways [
18,
19,
20]. Reduction of NO
2− to NO is facilitated in ischemic conditions [
19,
21], suggesting that this pathway of NO generation may protect the tissues from metabolic stress. Inorganic NO
3− supplements, such as concentrated beetroot juice, have been shown to acutely improve exercise tolerance in older adults [
22] and in patients with peripheral arterial disease [
23,
24]. Vascular mechanisms may contribute to this enhancement in exercise tolerance [
25,
26], as NO
3− supplements have been shown to induce a temporary reduction in blood pressure [
27] and arterial stiffness [
28,
29,
30] in older adults. Increased NO bioavailability through NO
3− supplementation may promote vessel dilation [
31], increasing blood flow, which might contribute to improved exercise tolerance in older adults.
Flow mediated dilatation (FMD) is a well-established measure of endothelial function [
32] where age-related decreases are associated with increased cardiovascular risk [
33,
34]. There are some studies supporting the link between NO
3− supplementation and increased FMD in the brachial artery of the arm in older adults [
28,
29,
30,
35]. While FMD is most commonly assessed at the arm, arm-FMD may not provide insight into the age-specific impairments that manifest in the legs. There is evidence that vascular impairments with ageing are limb specific [
11,
36] and arm-FMD is not predictive of leg-FMD [
37]. Moreover, brachial FMD does not reflect disease severity in lower-limb peripheral arterial disease [
38], although there may be a common link to elevated levels of reactive oxygen species [
39]. Identifying whether dietary NO
3− has an effect on FMD in the legs will help to establish whether dietary NO
3− may improve exercise tolerance through a mechanism that involves improved endothelial function.
Measurement of the hyperaemic response to passive leg movement provides an alternative index of vascular function and one that is specific to the lower limbs. While FMD, described above, is a measure of conduit artery function, the blood flow response during passive leg movement likely reflects endothelial function at the level of the downstream arterioles. Passive leg movement delivers mechanical stimulation without altering metabolic demand [
40], initiating a hyperaemic response that is highly dependent on NO bioavailability [
41,
42]. Consistent with this, passive leg movement hyperaemia declines with age [
43], is greater in those who exercise regularly compared to sedentary individuals [
44], and is diminished in cardiovascular disease [
13,
41]. Passive leg movement hyperaemia provides an opportunity to test the influence of NO
3− supplementation on NO-dependent leg blood flow in older adults.
The aim of this study was to determine whether an acute dose of inorganic dietary NO3− would improve femoral artery FMD, increase passive leg movement hyperaemia, and reduce arterial stiffness. Improvements in these parameters would suggest enhanced vascular function as a mechanism that contributes to improvements in exercise tolerance in older adults following dietary NO3− supplementation.
4. Discussion
The primary aim of this placebo-controlled, double-blind, cross-over design study was to determine whether NO3− supplementation would improve vascular function in older adults. The main findings are that an acute dose of dietary NO3− increased plasma NO2−, suggesting increased NO bioavailability, and this was associated with enhanced lower limb FMD and improved (lower) pressure augmentation index. However, dietary NO3− did not alter pulse wave velocity or the hyperaemic response to passive leg movement.
Our interest in the impact of NO
3− supplementation on vascular function in the leg began with the observation that NO
3− supplementation was associated with a decrease in blood pressure [
62] and an improvement in exercise tolerance in older adults [
22], and in patients with peripheral arterial disease [
23,
24], heart failure with preserved ejection fraction [
47], and stable angina [
63]. It was proposed that dietary NO
3− may enhance plasma NO
2−, which may increase NO bioavailability [
31]. Nitric oxide may then reduce blood pressure by improving vessel dilation and reducing arterial stiffness [
62] and may also contribute to enhanced exercise tolerance [
22] by increasing blood flow.
All participants responded to NO
3− supplementation with an increase in blood plasma NO
3− and NO
2− while there was no change in plasma NO
3− or NO
2− during the placebo trial. The link between NO
3− ingestion, circulating plasma NO
2−, and NO bioavailability has been clearly defined [
31], but individual responses can vary widely due to factors such as source and dose of NO
3−, dietary restriction prior to or during the experiment, oral microbiome and oral hygiene, medication and supplement use, and fitness status [
17,
64,
65]. We considered these factors in our study design and our participants showed an average 8.6-fold increase in circulating plasma NO
2− following NO
3− supplementation. This increase is similar in magnitude to other well-controlled studies investigating NO
3− supplementation in older adults [
23,
66] and indicates NO bioavailability was likely enhanced.
Our results demonstrate that FMD in the superficial femoral artery increased following NO
3− supplementation. Four previous studies with varied NO
3− doses (180–397 mg·day
−1 and 9.3 mg·kg body weight
−1), sources (beetroot juice, spinach, and NaNO
3), and treatment schedules (acute vs. ongoing) have indicated that NO
3− supplementation can increase brachial artery FMD in older adults [
28,
29,
30,
35]. A point of difference for the present study is that FMD was measured in the superficial femoral artery of participants, as opposed to the arm, as this site is likely to be of greater relevance to walking capacity and exercise tolerance. Additionally, the effect of age is not uniform across the cardiovascular system [
11,
12,
37], and upper limb FMD is not predictive of FMD in the lower limbs [
37]. Differences in the FMD response between upper and lower limbs may be related to differences in vessel size [
59], the thickness of the vessel walls [
67], or it may reflect the increased vulnerability of the lower limbs to the detrimental effects of aging and cardiovascular disease [
11,
12,
36]. For the first time, our study establishes that dietary NO
3− improves FMD in the superficial femoral artery, reflecting an enhanced endothelial function in the legs of older adults.
Previous studies of the effect of NO
3− supplementation on brachial FMD in older adults report inconsistent findings, with some indicating an increase as noted above [
28,
29,
30,
35], while others report no effect [
23,
68]. Similar to our study, the previous studies demonstrating a positive effect of NO
3− on FMD included participants who did not take medication and were free from disease [
28,
30,
35,
69]. The two studies reporting no change in brachial FMD following NO
3− supplementation included participants who were taking anti-hypertensive and anti-platelet medications. Anti-hypertensive medications are likely to have a vasodilating effect [
70] which may mask any effect of NO
3− supplementation. Another possible explanation for the null findings is that these two studies included participants who had been diagnosed with peripheral arterial disease [
23] and type II diabetes [
68]. Low brachial FMD is a sub-clinical marker for cardiovascular disease risk [
32,
34], which does not appear to change with improvements in walking distance for PAD patients [
23], however there is evidence to suggest that FMD in the leg, rather than the arm, may be a more sensitive measure in this patient group [
24,
71]. Regardless, the current mix of findings is promising, but indicates a need for further investigation into the therapeutic benefits of dietary NO
3− supplementation for individuals with cardiovascular disease.
Reactive hyperaemia did not increase following NO
3− supplementation, suggesting there was no change in vasodilation of arterioles downstream from the site of cuff occlusion. Importantly, this indicates that the increase in FMD was not a result of increased shear stress. One explanation for this finding is the dampening effect that dietary NO
3− may have on the abundance of reactive oxygen species [
72,
73]. Elevated production of reactive oxygen species is detrimental to vascular function, primarily because it leads to a loss in bioactive NO [
74,
75]. It is possible that NO
3− supplementation enhanced the antioxidant defence system [
76,
77], reducing the rate of reactive oxygen species production. A decrease in reactive oxygen species may have facilitated an increase in FMD, the magnitude of which might reflect eNOS-derived NO [
32], rather than NO produced via the NO
3−–NO
2−–NO pathway. This theory is supported by evidence of improved FMD following antioxidant treatments, such as vitamin E [
78,
79] and vitamin B3 (niacin) [
80]. Dietary NO
3− from vegetables such as beetroot may reduce oxidative stress more than individual vitamins or minerals because it may simultaneously restore redox imbalance and enhance NO generation [
72]. This proposal warrants further exploration.
Passive leg movement hyperaemia, a recently established measure of NO-dependent vascular function [
41,
42], did not change following NO
3− supplementation. Passive leg movement hyperaemia is measured at the common femoral artery, which typically does not dilate during this assessment, and the hyperaemic response is understood to reflect downstream arteriole endothelium-dependent flow regulation [
55]. Passive leg movement studies have used pharmacological NOS blockade to demonstrate an essential role of NO in the hyperaemic response [
43,
81], but it is interesting that NOS blockade only suppressed hyperaemia in young participants. The null response in older participants with both NOS blockade and NO
3− supplementation suggests that passive leg movement hyperaemia may not be a sensitive test for fluctuations in NO bioavailability in older adults. Collectively, our findings of increased FMD, unchanged reactive hyperaemia, and unchanged passive leg movement hyperaemia, indicate that, in the leg, conduit artery function is sensitive to a dose of dietary NO
3−, while downstream arteriole flow regulation is not. While the increase in FMD following NO
3− supplementation indicates an improvement in lower limb endothelial function, the lack of change in reactive hyperaemia and passive leg movement hyperaemia raises questions about the functional relevance of this finding. The impact of dietary NO
3− on femoral blood flow during exercise has not been described in older adults. Given that conditions of low oxygen tension and low pH that may be observed during exercise promote reduction of NO
2− to NO, future studies should investigate whether exercise blood flow responses are augmented with dietary NO
3−.
Another explanation for the absence of an effect of NO
3− supplementation on passive leg movement hyperaemia is insufficient stimulation. Studies exploring NO
3− supplementation in hypoxia demonstrate that the conversion of NO
2− to NO is facilitated by perturbations to homeostasis, such as a reduction in oxygen tension [
21,
82,
83]. In the present study, during passive leg movement, participants were instructed to maintain relaxed muscles. Passive leg movement causes the muscle tissues to stretch without registering EMG activity or increasing muscle oxygen uptake [
40,
41,
84,
85], thereby not imposing any metabolic stress. The value of passive leg movement as an experimental model is that it avoids activation of other vasodilating systems associated with exercise, such as prostacyclin [
84,
86], revealing a blood flow response that reflects NO bioavailability. However, in our study, it is possible that elevated plasma NO
2− did not increase the hyperaemic response to passive leg movement because metabolic stress is necessary to activate mechanisms that produce NO from NO
2−.
Resting leg blood flow, assessed immediately prior to the onset of passive leg movement, was similar post-NO3− and pre- and post-placebo, however resting flow was lower pre-NO3−. Trial order was randomised for each participant and this measure was taken prior to ingestion of NO3−. There was no reason that we can identify to explain why leg blood flow was lower pre-NO3−.
Augmentation index tended to be lower in the afternoon during both the NO
3− and placebo trials, although the difference only reached significance following administration of dietary NO
3−. The time-related effect may reflect a redistribution of blood volume and reduced cardiac preload [
87] or there may be a diurnal effect in augmentation index. We suggest time of day should be standardised for this measure. Stiffening of arterial vessels is a risk factor for cardiovascular disease [
88,
89], as age-related stiffening in the peripheral vessels can cause reflected waveforms to meet outgoing pulse waves closer to the heart with higher pressure, impeding blood flow and putting strain on the left ventricle [
90]. For this reason, it is advantageous to reduce downstream resistance in older adults [
91,
92] and a reduction in the augmentation index after a dose of dietary NO
3− suggests a temporary cardiovascular benefit. Endothelial function and arterial stiffness are related [
90] and measures of FMD and augmentation index have previously been shown to be associated [
93]. The fact that both improved in the present study suggests that both may be sensitive to enhanced NO bioavailability, however FMD and augmentation index were not correlated. Lower augmentation index indicates an improvement in peripheral vessel compliance and this finding agrees with other studies that have measured augmentation index following NO
3− supplementation in older adults [
28,
29,
30].
Pulse wave velocity was unchanged by a dose of dietary NO
3−. This concurs with some [
94,
95,
96] but not all reports [
28,
29,
30]. Systolic and diastolic blood pressures were also unchanged following NO
3− supplementation. There are equivocal reports in the literature regarding whether dietary NO
3− reduces blood pressure in older adults [
27,
97]. There are many factors that influence blood pressure, including structural properties of vessels, neural input, and local vasodilator and constrictor regulation throughout the vascular network [
98]. However, the finding of no change in pulse wave velocity is consistent with the absence of any change in blood pressure in this study.
Limitations
Participant use of medication for cardiovascular conditions may mask the effect of NO
3− supplementation. Some participants in our study were taking low dose statins and, in addition to lowering cholesterol, statins may improve endothelial NOS expression and improve baseline NO bioavailability [
99]. The fact that baseline plasma NO
2− was low, even among those who were taking statins, suggests low NO status irrespective of medication use. During the study, there were no changes to the use of statins, or schedule or timing of dose for any individual, and experiment trials were carried out on the same day and time, separated by one week. Further, the cross-over design enabled participants to act as their own controls.
Sex differences have been noted in the literature regarding the effectiveness of dietary NO
3− supplementation [
100,
101,
102,
103]. There is evidence of greater circulating NO
2− in females, which is associated with lower blood pressure [
100]. This is consistent with demographic studies indicating that blood pressure is lower for post-menopausal women than age-matched men [
104] and it is also supported by evidence of increased NO production in women [
105]. It has been proposed that elevated NO
2− in females leads to a saturation effect that may reduce sensitivity to NO
3− supplementation compared to men [
106]. This investigation involved male participants and may not be generalisable to females. Future study is needed to confirm these findings in female participants.