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N.S. Bryan, J. Loscalzo (eds.), Nitrite and Nitrate in Human Health and Disease, Nutrition and Health,
DOI 10.1007/978-3-319-46189-2_18, © Springer International Publishing AG 2017
Keywords Vascular function • Physical function • Cognitive function • Oxidative stress • Inflammation
Introduction
Advancing age is associated with declines in physiological function and is the leading risk factor for
the majority of chronic degenerative diseases in modern societies [1]. This fact, combined with the
rapidly changing demographics of aging and record number of older adults, projects unprecedented
levels of clinical disease, disability, and health care burden in the near future [2–4]. As such, it is
imperative to identify the mechanisms underlying age-related physiological dysfunction and to
Chapter 18
Nitrate and Nitrite in Aging and Age-Related Disease
Lawrence C. Johnson, Allison E. DeVan, Jamie N. Justice, and Douglas R. Seals
L.C. Johnson, M.S.
Department of Integrative Physiology, University of Colorado Boulder, 354 UCB, Boulder, CO, 80309, USA
A.E. DeVan, Ph.D.
Medical College of Wisconsin, Cardiovascular Center, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA
J.N. Justice, Ph.D.
Department of Internal Medicine—Geriatrics, Wake Forest School of Medicine,
1 Medical Center Blvd., Winston-Salem, NC, 27012, USA
D.R. Seals, Ph.D. (*)
Department of Integrative Physiology, University of Colorado Boulder, 354 UCB, Boulder, CO, 80309, USA
e-mail: seals@colorado.edu
Key Points
• Advancing age is associated with declines in physiological function and is the leading risk factor
for the majority of chronic degenerative diseases in modern societies.
• Central to age-associated declines in physiological function is a reduction in the endogenous pro-
duction and bioavailability of the ubiquitous signaling molecule nitric oxide.
• Supplementation with nitrate and/or nitrite boosts nitric oxide bioavailability and improves physi-
ological function in the presence of aging.
• Evidence suggests that nitrate and/or nitrite supplementation may hold promise as therapies to
preserve and/or improve physiological function and reduce the risk of chronic degenerative dis-
eases in middle-aged and older populations.
260
develop therapeutic treatments that can prevent or slow these processes; delay the onset of functional
limitations, disability, and chronic disease; and extend quality of life to later ages. Indeed, increasing
health span, or the number of years free of major functional impairment and clinical disease, is now
recognized as a top priority in biomedical research [4–6].
Age-Associated Declines in Nitric Oxide
Nitric oxide (NO) is a gaseous signaling molecule critical to the regulation of numerous physiological
processes, and its presence is essential to the preservation of physiological function and health with
advancing age. NO is produced via two main biological pathways in vivo. First discovered was the
l
-arginine–NO-synthase pathway in which a stimulus causes nitric oxide synthase (NOS) enzymes to
catalyze the reaction of
l
-arginine with oxygen to produce NO [7]. More recently, a second pathway
has been elucidated by which NO can be produced through the reduction of nitrate and nitrite by vari-
ous reducing mechanisms, including nitrate and nitrite reductases [8, 9]. NO has a very short half-life,
as the molecule is quickly oxidized to nitrite and, subsequently, nitrate. Both nitrite and nitrate are
unique in that they can act as stable circulating storage forms of NO that can be rapidly reduced to
restore NO levels in vivo.
NO bioavailability decreases with age, incurring deleterious effects on the systems that require NO
to maintain proper signaling and function. The reasons for the age-associated decrease in NO bio-
availability are multifactorial, but evidence suggests that an age-related increase in oxidative stress is
the primary cause [8, 10]. Oxidative stress is defined as an imbalance in reactive oxygen species
(ROS) relative to antioxidant defenses (Fig. 18.1) [11]. Oxidative stress reduces NO bioavailability
with aging via several mechanisms. Excessive age-related production of superoxide, a prominent
ROS, reacts readily with NO to form peroxynitrite and, in so doing, directly reduces the abundance of
Fig. 18.1 Potential mechanisms by which advancing age leads to physiological dysfunction and the beneficial effects
of nitrate and nitrite supplementation on these processes. ROS reactive oxygen species, NO nitric oxide
L.C. Johnson et al.
261
NO [12]. Excessive superoxide also dysregulates (“uncouples”) NOS enzymes by oxidizing the
essential cofactor for NO production, tetrahydrobiopterin (BH4), resulting in further superoxide pro-
duction and reduced NO synthesis [13].
In addition, age-associated oxidative stress is created and maintained by increased expression and
activity of pro-oxidant enzymes (e.g., NADPH oxidase) in the absence of compensatory up-regula-
tion of endogenous antioxidant enzymes [14–16]. More recently, dysregulated mitochondria have
been implicated in excess production of ROS with aging [17, 18]. Chronic, low-grade inflammation
also develops with advancing age, reinforces oxidative stress, and contributes to systemic physiologi-
cal dysfunction [19–22] even in the absence of clinical disease. Given the earlier, lifestyle and phar-
macological strategies that restore NO bioavailability with aging, perhaps in part by inhibiting these
pro-oxidant processes and inflammation, hold the promise of enhancing and maintaining physiologi-
cal function with aging..
Nitrate and Nitrite Supplementation Improves Physiological Function
Nitrate and nitrite supplementation-based therapies are effective in increasing the concentrations of
these NO precursors in vivo [23–25]. Such treatments can be successfully administered via several
different methods, including inhalation, intravenous or intra-arterial infusion, topical application, or
most commonly, oral ingestion. Circulating and tissue concentrations of nitrate and nitrite have been
boosted by oral consumption of salts (e.g., NaNO3−, KNO3, NaNO2−) or green leafy or root vegetables
(e.g., spinach, beetroot juice). These modes of delivery provide a pool of systemically available nitrate
and nitrite, substrates with which to produce NO independent of the endogenous
l
-arginine–
NO-synthase pathway in both healthy individuals and those suffering from age-related disease states
associated with decreased NO bioavailability. The following sections review changes in selective
physiological functions with aging, the potential mechanisms by which age-related changes in NO
bioavailability or signaling may affect these functions, and the current evidence from preclinical and
clinical trials that suggest efficacy of nitrate or nitrite supplementation on age-related physiological
dysfunction (Fig. 18.2).
Cardiovascular Diseases
Age is the major risk factor for cardiovascular diseases (CVD), as >90 % of all deaths from CVD
occur in older adults above 55 years of age [26]. The increased risk of CVD with aging is due in large
part to adverse changes occurring in arteries associated with vascular dysfunction [10, 27]. Among
these changes, a decline in NO bioavailability is a critical event [10, 28]. NO is produced within the
vascular endothelium and diffuses to the vascular smooth muscle, providing a powerful vasodilatory
signal that adjusts the diameter of the vessel to accommodate changes in blood flow. In aging as well
as pathological states in which NO bioavailability is low, this signaling pattern is disrupted, creating
an environment conducive to endothelial dysfunction and stiffening of the large elastic arteries, two
primary contributors to the increased risk of CVD in middle-aged and older adults [29, 30]. Resulting
from these changes to the large elastic arteries, sensitive organ systems such as the kidney, brain, and
liver can be damaged owing to alterations in blood flow or the elevated pulsatile hemodynamics cre-
ated and directed to delicate microvascular systems in these organs [31, 32]. Therefore, restoring the
age-related decline in NO bioavailability and thereby decreasing large elastic artery stiffness and
improving endothelial function may serve to reduce the prevalence of CVD and preserve function in
multiple organ systems.
18 Nitrate and Nitrite in Aging and Age-Related Disease
262
Endothelial Dysfunction
The endothelium is a single cell layer that lines the vasculature at the interface of the arterial wall and
the lumen of the vessel, in direct contact with the flow of blood. Via release of NO, the endothelium
exerts a major influence on vasodilation, inhibition or activation of smooth muscle proliferation, and
inflammatory processes [33]. As such, the endothelium plays an important role in maintaining vascu-
lar health [34].
Advancing age is associated with endothelial dysfunction, a pathological state of the endothelium
in which there is an imbalance between vasodilating and vasoconstricting factors [33]. Additionally,
a dysfunctional endothelium induces smooth muscle cell proliferation, platelet activation and adhe-
sion, and inflammation, resulting in a phenotype that further drives vascular pathology and declines
in function [35]. A primary hallmark of age-related endothelial dysfunction is the decreased synthesis
and/or bioavailability of NO [28].
The ability of nitrate and nitrite therapies to produce NO through a pathway independent of eNOS
makes them attractive therapies for restoring vascular function [36, 37]. Indeed, the vasodilating abili-
ties of nitrate and nitrite have been known for decades [38–41], but due to safety concerns and mis-
conceptions regarding their use, these compounds have not been tested for efficacy in treating vascular
aging until recently. Early studies in humans focused on the acute effects of infused nitrate or nitrite
on vascular function as measured by changes in forearm blood flow. Intravascular infusions of sodium
nitrite acutely increased forearm blood flow in both young and middle-aged healthy adults [42–44],
older adults [45], and some clinical populations [46, 47]. Other studies directly assessed endothelial-
dependent dilation (EDD), a well-established measure of endothelial function, and demonstrated the
acute beneficial effects of nitrate or nitrite administration [48–51].
Fig. 18.2 Nitrate and nitrite supplementation counteract age-associated functional declines and improve health
outcomes. I/R ischemia/reperfusion
L.C. Johnson et al.
263
To address the chronic effects of sodium nitrite, vascular endothelial function was assessed via
EDD in response to increasing doses of acetylcholine, a compound used for pharmacological activa-
tion of eNOS and production of NO in mouse models of primary aging. Compared with young ani-
mals, arteries from old mice displayed impaired vasodilation in response to acetylcholine. However,
3 weeks of sodium nitrite [52] or 8 weeks of sodium nitrate [53] administered in the drinking water
restored EDD in old mice to levels seen in young animals. Pharmacological investigation of the
mechanisms by which sodium nitrite improves EDD revealed that the effects on vascular function
were mediated by increased NO bioavailability [52]. The effects of sodium nitrate were confirmed in
a human trial in which 4 weeks of sodium nitrate supplementation improved EDD in healthy older
adults with elevated risk factors for CVD [54]. Results of a recent study in which sodium nitrite was
orally administered for 10 weeks also showed improvements in EDD in healthy middle-aged and
older adults [55]. These findings indicate that acute and chronic nitrate and nitrite supplementation is
well tolerated, and that the effects of these therapies are sufficient to improve age-associated declines
in NO bioavailability and resulting impairments in endothelial function.
Large Elastic Artery Stiffness
The large elastic arteries play an important role in regulating blood flow and pressure throughout the
systemic circulation. The elastic properties of the large arteries work to dampen the pressure waves
associated with the ejection of blood from the heart and protect sensitive organs from high pulsatile
flows. Permitting this phenomenon in healthy arteries are elastin, the protein component of arterial
walls that allows for increased elasticity and compliance; and collagen, a nonelastic protein compo-
nent utilized for structural integrity under high-pressure loads. A proper ratio of structural compo-
nents contributes to a healthy, compliant vessel.
With primary aging, as well as in the presence of certain disease states, there is a gradual increase
in large artery stiffness. The stiffening of the large elastic arteries is facilitated through the structural
remodeling of the wall of the large arteries [56]. Fragmentation of elastin and an increase in collagen
deposition contribute to reduced arterial compliance and stiffen the large elastic arteries [57]. Lastly,
the formation of advanced glycation end-products (AGEs) also contributes to the stiffening process of
large elastic arteries by cross-linking structural proteins in the extracellular matrix [58]. These changes
occur in settings of both healthy aging and disease and have been associated with increased risk of
cardiovascular events in humans [30, 56, 59–61].
Trials to test the efficacy of using nitrate and nitrite therapies as interventions to improve large
elastic artery stiffness are relatively recent. In a preclinical study, increased arterial stiffness was
observed in healthy old mice compared to young controls. Although there were no effects on elastin
or collagen content, treatment with sodium nitrite reversed the age-associated increase in stiffness
through reduced abundance of AGEs in the arterial wall [52, 62]. Supporting the effects in mice, acute
and chronic supplementation with nitrates reduces arterial stiffness in healthy and diseased adults.
Specifically, central arterial stiffness was reduced in response to an acute dose of potassium nitrate
[63], intra-arterial infusion of sodium nitrite [64], or dietary nitrate from beetroot juice [65] in young
healthy adults, and/or healthy controls and patients with hypertension. In addition, within 30 min,
arterial compliance was improved in hypertensive patients after a single acute dose of a nitrite con-
taining lozenge [66]. Consistent with these observations, chronic supplementation of nitrite and
nitrate also improved measures of arterial stiffness. Healthy middle-aged and older adults given
sodium nitrite supplementation for 10 weeks demonstrated increased carotid artery compliance and
decreased β-stiffness index without alterations in central or peripheral blood pressure [55].
Additionally, 4 weeks of beetroot juice or sodium nitrate dissolved in water decreased aortic pulse
wave velocity, the gold-standard measure of aortic stiffness, in patients with hypertension [67] and
18 Nitrate and Nitrite in Aging and Age-Related Disease
264
older adults at risk for developing CVD [54]. Overall, there is some evidence that nitrate and nitrite
may have beneficial effects in populations with increased baseline large elastic artery stiffness, but
much more work is needed to demonstrate this benefit definitively.
Blood Pressure
Older adults are at high risk for the development of hypertension [68], and the literature currently
suggests that nitrate and nitrite treatment may be effective at reducing blood pressure. Several studies
have reported that acute nitrite administration reduces blood pressure in animal models of hyperten-
sion [69], in clinical studies of healthy adults [70], in addition to patients with diabetes [71], pre-
hypertension [72], untreated hypertension [66], and urea cycle disorders [73]. Studies involving
nitrate administration have yielded more varied results. On the one hand, a meta-analysis found that
nitrate supplementation for 7 to 21 days did not have a pooled systemic effect on ambulatory blood
pressures [74]. Additionally, patients with diabetes and those previously treated for hypertension dis-
played no further reduction in blood pressures after at least 1 week of nitrate supplementation, despite
experiencing increases in circulating nitrate and nitrite [75, 76].On the other hand, both acute and
chronic nitrate (up to 15 days) supplementation has been reported to reduce blood pressures in healthy
adults and those with moderate risk factors for, but no history of, CVD [50, 77–79]. Moreover, in a
recent double-blinded, placebo-controlled randomized trial, dietary nitrate was shown to provide sus-
tained blood pressure lowering in hypertensive patients [67]. Daily dietary nitrate supplementation
was associated with both systolic and diastolic blood pressure reductions as measured by three differ-
ent methods. Blood pressure measured at home was significantly reduced by 8.1/3.8 mmHg (P<0.001
and P<0.01), while blood pressure measured in the clinic experienced a mean reduction of 7.7/2.4
mmHg (P<0.001 and P=0.050). Importantly, 24-h ambulatory blood pressure declined by 7.7/5.2
mmHg (P<0.001 for both) during the 4 week intervention period in the absence of tachyphylaxis.
Vascular function also improved after dietary nitrate consumption, as arterial stiffness was reduced by
0.59 m/s (P < 0.01) while endothelial function was significantly improved by ≈ 20 % (P < 0.001) with
no changes in the placebo group. Supporting these findings is evidence that 4 weeks of daily dietary
inorganic nitrate supplementation lowers systolic blood pressure, reduces vascular stiffness, and
improves endothelial function in an elderly population with moderately increased cardiovascular dis-
ease risk [57]. More studies will be needed to determine how effective inorganic nitrite and nitrate
truly are in the treatment of hypertension. Thus, presently there is no scientific consensus as to the
effects of nitrate and nitrite on age-associated hypertension, but this remains a promising avenue of
investigation.
Myocardium
With advancing age, key structural changes, such as myocyte hypertrophy, diminished myocyte num-
ber, and increased connective tissue, contribute to higher levels of myocardial stiffness, impaired con-
tractile response, and other alterations in excitation–contraction coupling that diminish myocardial
function [27]. The effects of nitrate or nitrite therapy on primary cardiac aging are currently unknown,
but results from numerous studies have demonstrated the beneficial effects of nitrite or nitrate admin-
istration for reducing myocardial ischemia/reperfusion injury in preclinical models [23, 80–83]. The
L.C. Johnson et al.
265
protection afforded by nitrate and nitrite is attributed, at least in part, to their roles as physiological
stores of NO and modulators of mitochondrial function. Confirmation of these findings in humans is
limited at this time. Currently, only two clinical trials have addressed this issue in patients with
ST-segment elevation myocardial infarction (STEMI) undergoing percutaneous coronary intervention,
with conflicting results that could be due, in part, to differing doses and routes of administration.
Intravenous infusion of sodium nitrite did not reduce myocardial infarct size or alter secondary end-
points in one study [84], whereas infusion of a higher dose of nitrite directly into the coronary arteries
was associated with a lower number of major adverse cardiac events and a higher myocardial salvage
index at 1-year follow-up [85]. In a subgroup analysis of patients treated with thrombolysis, nitrite also
reduced infarct size [85], and a phase III trial is presently underway.
Another target for therapy with nitrate or nitrite administration is heart failure. Patients with heart
failure have low cyclic guanosine monophosphate (cGMP) content and low cGMP-dependent protein
kinase (PKG) activity in their myocardium, which contribute to the development of myocardial hyper-
trophy, increased passive stiffness, and delayed myocardial relaxation [86]. Enhanced NO bioavail-
ability improves cGMP/PKG signaling as well as vasodilation in the coronary and peripheral
circulations, augmenting perfusion of the heart and sub-endocardium [47, 87]. Consistent with such
benefits, a recent pilot study found that a single dose of nitrate-rich beetroot juice improved exercise
capacity in patients with heart failure [88]. Similarly, infusion of sodium nitrite markedly improved
forearm blood flow in patients with congestive heart failure [47] and improved functional cardiac
responses during a dobutamine stress echocardiogram in patients with inducible myocardial ischemia
[49]. Taken together, the results from these initial studies support the continued investigation of nitrate
and nitrite for the management of heart failure, and perhaps myocardial infarction.
Dysfunction of Other Physiological Systems
In addition to their promise as therapies for CVD, nitrate and nitrite may also have beneficial effects
on other domains of physiological function and health with aging. Because NO is a universal signal-
ing molecule, it affects multiple organ systems and integrative functions. By increasing NO bioavail-
ability and its other NO-independent actions, nitrate and nitrite therapies may attenuate age-related
declines in cognitive and physical functions.
Cognitive Function
Cognitive decline and dementias directly related to age have been well documented. Advancing age
preferentially impairs select domains of cognitive function, most notably memory and executive
functioning, i.e., the processes that support strategic organization required for complex, goal-oriented
tasks [89]. A number of pathophysiological changes occur in aging that are candidates for causing
age-associated executive and memory difficulties, namely, neuronal atrophy, white matter abnormali-
ties, and neurochemical changes within the brain [90, 91]. Indeed, 65 % of nondemented older adults
(>75 years) show white matter abnormalities [92], which are consistent with general atrophy and loss
in brain volume [93]. Small infarcts are also prevalent, as are white matter lesions thought to result
from vascular disorders, such as small vessel disease [94, 95]. Importantly, these white matter lesions
are strongly associated with, and predictive of, declines in both executive function and memory [90],
even in nondemented older adults [96, 97]. Several lines of evidence suggest that age-associated vas-
cular dysfunction may be an important mechanism underlying executive and memory impairment
[98], and that oxidative stress and NO deficiency are key underlying factors [98, 99].
18 Nitrate and Nitrite in Aging and Age-Related Disease
266
Accumulating findings indicate that NO plays an important role in the preservation of cognitive
health with aging. As a multifunctional messenger molecule, NO has a prominent role in both regu-
lation of cerebral blood flow and cell-to-cell communication in the brain. Through its vasodilatory
effects, NO contributes to the regulation of cerebral perfusion [100]. Numerous theories now posit
that a reduction in NO bioavailability, whether from advanced age or a perfusion-lowering disease
condition, results in hemodynamic microcirculatory insufficiency [98]. If impaired perfusion per-
sists below a key threshold, referred to as the “critically attained threshold of cerebral hypoperfu-
sion” (CATCH), it can lead to a restricted energy state that may destabilize neurons, synapses, and
neurotransmission, and ultimately affect cognitive function [101, 102]. An additional mechanism by
which NO may link vascular function to cognition is through regulation of neurovascular coupling,
which is the time- and regional-dependent connectivity between local neural activity and subsequent
changes in cerebral blood flow. For example, when neurons and glia generate signals, this initiates a
coordinated cascade of vascular events, ultimately dependent on NO to produce vasodilation to the
specific area of activation in a timely manner. Reduced NO bioavailability and signaling has been
implicated in diffuse and disrupted coupling in which the cerebral blood flow is no longer matched
to the metabolic requirements of the tissue [103].
Evidence supports the potential for administration of nitrates, nitrites, and NO donors to improve
NO bioavailability and improve neuronal/cognitive function with age, although this has not been
thoroughly vetted in clinical trials of older adults. A preclinical trial established that old rats display
impaired retention, object recognition, and discrimination capabilities compared to young animals;
yet, old animals administered molsidomine, a direct NO donor, showed complete restoration of func-
tion to that of young rats in retention and discrimination abilities [104]. The effects of boosting NO
on learning and memory in rodents have been reviewed extensively elsewhere [105], and the general
consensus from these investigations is that proper NO signaling improves behaviors reliant on cogni-
tion, while the inhibition of NO synthesis induces cognitive impairment. In contrast to this preclini-
cal evidence, little work has been performed examining the effects of NO boosting agents in older
men and women. In one of only few available trials performed in healthy middle-aged and older
adults, short-term (3-day) supplementation with dietary nitrate in the form of beetroot juice failed to
induce improvements in cognition as determined by a computerized battery of tests, although coad-
ministration of sodium nitrate with the carbonic anhydrase inhibitor acetazolamide did improve
cerebral blood flow to visual stimuli in healthy males [106]. However, nonhuman primate models of
stroke have shown nitrite can cross the blood–brain barrier and inhibit cerebral vasospasm, support-
ing its possible efficacy for improving age-associated brain health [107]. Moreover, 10 weeks of
sodium nitrite supplementation improved performance on Trail Making Tests A and B, measures of
executive function, in healthy middle-aged and older adults [108]. Overall, these results suggest that
NO plays a significant role in learning and memory mechanisms affected by increasing age, and
demonstrate the necessity of NO as a signaling molecule and vasoactive regulator in the domain of
cognitive function, although the length and type of supplementation may be key to inducing clini-
cally important improvements.
Physical Function
The ability to perform physical tasks is critically important to maintaining overall functional capacity
[109–111], and physical function has emerged as a predictor of morbidity and mortality in older adults
[111–113]. Although no single cause has emerged as being responsible for the onset of deficits in
physical function with advanced age, many interconnected factors contribute to this inevitable decline
[114]. The age-related physical disablement process begins with physiological impairments such as
motor neuron loss and subsequent remodeling, impaired transmission at the neuromuscular junction,
L.C. Johnson et al.
267
increased skeletal muscle excitation–contraction uncoupling, loss of mitochondrial efficiency,
impaired vascular coupling, and eventual skeletal muscle atrophy [109, 114–117]. These physiologi-
cal impairments lead first to observable deficits in muscle power and strength, then functional limita-
tions such as reduced walking speed or ability to rise from a chair, and eventually culminate in
disability and loss of independence [109, 118].
Many points in this sequence of physiological events contributing to functional limitation and age-
related physical disability may be mediated by NO bioavailability and signaling, although little work
has been performed linking NO to physical function in primary aging. First, as described previously,
NO has beneficial effects contributing to neuroprotection which could theoretically inhibit the mor-
phological loss of motor neurons and loss of axonal transmission of neural signals that are a hallmark
of primary aging and a leading contributor to subsequent impairment, though this has not been tested
empirically. Second, NO has known antioxidant properties that could limit excitation–contraction
uncoupling, which is largely mediated by the oxidative modification of dihydropyridine receptor
(DHPR) in the electromechanical transduction step linking neural input to the release of Ca2+ intracel-
lularly to cause cross-bridge binding and force production [116]. Previous work in young animals
supports this mechanistic action as boosting NO through dietary nitrate increases DHPR expression,
intracellular Ca2+ release, and force production in skeletal muscle [119]. Third, as reviewed elsewhere
in this book, NO is critically important in mitochondrial regulation and improves mitochondrial effi-
ciency in young adults, which is accompanied by increased work rate and exercise tolerance [120].
Finally, vascular function is significantly related to physical function in older adults, including muscle
fiber type and morphology, muscle power and performance in activities of daily living that require
balance, upper and lower body strength, flexibility, balance, and coordination [121, 122].
The ability of nitrate and nitrite to modulate vascular function beneficially, along with its positive
antioxidant and mitochondrial effects, provides optimism that nitrate and nitrite may restore physical
function in middle-aged and older adults; yet, few studies have tested the hypothesis that nitrate and
nitrite can improve measures of physical function with advanced age. In one such study, young and
old mice were assessed for grip strength, open-field distance, and rota-rod endurance [123]. Results
showed that old mice had deficits in these functional measures compared to young, and that 8 weeks
of sodium nitrite supplementation improved grip strength and open field distance while completely
restoring rota-rod endurance to that of young animals. Preclinical and clinical studies have also con-
firmed nitrate as an effective means to improve measures of physical performance, including mea-
sures of strength, exercise capacity, and endurance in young individuals as a result of increased NO
bioavailability [124–126]. Trials in older adults are few, but an early study investigating the role of
NO in physical function found that administration of
l
-arginine, a precursor of NO, improved mea-
sures of force production in postmenopausal women [127]. Although increasing NO bioavailability
with 3 days of oral nitrate supplementation through beetroot juice consumption was insufficient to
improve performance in a 6-min walk test in older adults [72], utilizing acute doses of nitrate has been
found to be beneficial for certain domains of physical function. Acute administration of beetroot juice
was shown to increase circulating nitrite levels in healthy older adults and significantly improve con-
tracting skeletal muscle blood flow during handgrip exercise under hypoxic conditions [128].
Furthermore, older patients with peripheral artery disease experience a decrease in blood pressure and
an improvement in exercise time prior to claudication pain after an acute dose of nitrate [129]. These
results support the potentially beneficial role of nitrate on exercise capacity and motor performance in
older populations, and particularly in groups with impaired skeletal muscle blood flow. In agreement
with these findings administering nitrate, a recent study demonstrated that 10 weeks of sodium nitrite
supplementation improves indices of balance, endurance, and muscle power in healthy middle-aged
and older adults free of disease and disability [108]. Further clinical trials with nitrate or nitrite are
needed to assess efficacy in a more comprehensive battery of physical function assessments in both
healthy older adults and patients with clinical disease.
18 Nitrate and Nitrite in Aging and Age-Related Disease
268
Injury and Disease
Research into the possible health benefits of nitrate and nitrite includes numerous other systems, tis-
sues, and conditions related to aging, including pulmonary and renal function and ischemia–reperfu-
sion injury. Preclinical models of pulmonary function show beneficial effects of nitrate and nitrite
therapies. Specifically, pulmonary hypertension is improved with nitrate and nitrite supplementation
in preclinical models [130], and clinical investigations confirm these effects in humans, with an acute
nitrite infusion reducing pulmonary pressures in states of hypoxia [44], and a single dose of dietary
nitrate improving exercise performance and blood pressure responses in patients with chronic obtru-
sive pulmonary disease [131].
Numerous studies have established nitrite as a protective treatment in the setting of ischemia–
reperfusion injury in multiple tissues and organs [23, 80, 132, 133]. Nitrite is effective at improving
outcomes after ischemia in skeletal muscle [134], kidneys [135], liver [136], and lungs [137].
Furthermore, renal function is favorably affected by nitrite in other compromised states, such as renal
injury under conditions of eNOS inhibition [138] and brain death-mediated renal injury [139]. Lastly,
nitrite is effective at improving function and reducing adverse outcomes in models of heart transplan-
tation [140]. Taken together, these investigations confirm that nitrate and nitrite therapies may have
efficacy in numerous pathophysiological states that affect older adults.
Mechanisms by Which Nitrate and Nitrite Improve or Preserve Function
with Advancing Age
Oxidative Stress
Aging is associated with increases in oxidative stress that can damage cellular components and induce
dysfunction in organs and systems, promoting disease [141–143]. For example, increased oxidative
stress within arteries has been shown to be a primary contributor to the development of arterial dys-
function with age [144–146]. Administration of sodium nitrite to old mice normalized nitrotyrosine
levels (a cellular marker of oxidative damage to proteins) to that of young animals, indicating a reduc-
tion in age-associated oxidative stress [52, 62]. Age-associated oxidative stress is driven by exces-
sively high levels of superoxide production, which was attenuated with sodium nitrite [52]. Nitrate
and nitrite supplementation also reduce oxidative stress in preclinical models of injury and disease.
Hypertension in both the presence and absence of compromised renal function, ischemia–reperfusion
injury, and cardiomyopathy are all associated with increased oxidative stress that is ameliorated with
nitrate or nitrite supplementation [65, 147–149].
Increasing age is associated with elevated concentrations and activity of the superoxide-generating
enzyme NADPH oxidase in multiple organ systems, including the vasculature [150–152]. NADPH
oxidase protein expression is higher in old compared to young aortas of mice, suggesting an age-
associated increase in NADPH oxidase abundance. Three weeks of sodium nitrite supplementation
reduced the abundance of NADPH oxidase in old mice to levels seen in the young, demonstrating the
ability of sodium nitrite to down-regulate the expression of this pro-oxidant enzyme [52]. To deter-
mine if functional changes are associated with altered enzymatic activity of NADPH oxidase, isolated
carotid arteries from aged animals were incubated with the NADPH oxidase inhibitor apocynin. Old
animals treated with apocynin had improved endothelial function, while no effect was seen in young
controls or old animals treated with sodium nitrite [52], indicating that age-associated increases of
NADPH oxidase and its activity inhibit EDD, and that sodium nitrite is successful in reversing these
L.C. Johnson et al.
269
effects. Similarly, pentaerythritol tetranitrate, an organic nitrate, reduces NADPH oxidase activity in
the cardiac tissues of diabetic rats [153]. Collectively, these results suggest that nitrate and nitrite are
effective in lowering oxidative stress by reducing the expression and activity of the pro-oxidant
enzyme NADPH oxidase.
Antioxidant Defenses
Endogenous antioxidant enzymes are primarily responsible for combating the increase in oxidative
stress. Antioxidant enzymes, such as superoxide dismutase (SOD), scavenge ROS in an attempt to
maintain oxidative homeostasis. Declines in antioxidant enzyme expression and activity contribute to
the development of oxidative stress with aging [154, 155]. SOD activity declines with aging in the
aortas of mice, and sodium nitrite supplementation restored SOD activity to levels observed in the
young animals [52]. Subsequent trials have confirmed the ability of nitrite to increase antioxidant
defenses in preclinical models of vascular hypertension, ischemia–reperfusion injury, renal injury,
and alcohol-induced liver injury [69, 139, 156–159]. A recent study in humans showed that sodium
nitrite can improve oxidative states associated with peripheral artery disease and diabetes by improv-
ing the GSH:GSSG ratio [160]. These investigations suggest nitrite supplementation may be an effec-
tive means to restore antioxidant defenses in aging and disease.
eNOS Uncoupling
An increase in eNOS uncoupling via decreased BH4 has been implicated in the decrease in NO bioavail-
ability. To understand the contribution of eNOS uncoupling to reduced EDD, carotid arteries from young
and old mice were incubated ex vivo with sepiapterin, a compound that restores BH4 bioavailability and
re-couples eNOS, dramatically increasing NO production. In old mice, sepiapterin improved EDD to
levels similar to young animals while having no effect in old mice administered sodium nitrite, suggesting
that eNOS uncoupling via BH4 deficiency is at least partially responsible for impaired EDD in old mice
[161]. Additionally, nitrite has been shown to improve EDD in hypercholesterolemic mice through main-
tenance of BH4/BH2 ratios [162]. Importantly, these results also support nitrite supplementation as a pos-
sible treatment to increase BH4 bioavailability, recouple eNOS, and increase the bioavailability of NO in
states characterized by eNOS uncoupling, including advanced age, and some diseases.
Mitochondrial Dysfunction
Recently, mitochondrial dysfunction has been identified as a potential mechanism underlying several
chronic diseases associated with aging, including CVD and diabetes [25, 163–165]. Age-associated
declines in mitochondrial function have been established in the vasculature of old mice and have been
implicated in the development of vascular dysfunction [166]. Studies investigating the role of NO on
mitochondrial function have found that low NO concentrations can cause impaired mitochondrial fit-
ness, including unfavorable mitochondrial remodeling and a decline in ATP production. Conversely,
evidence suggests that mitochondrial biogenesis and mitochondrial antioxidant enzyme expression
can be up-regulated in conditions of sufficient NO bioavailability [167–171].
The effects of nitrate supplementation on mitochondrial function were evaluated in a placebo-con-
trolled crossover study involving healthy humans. Independent of increases in mitochondrial content,
18 Nitrate and Nitrite in Aging and Age-Related Disease
270
nitrate supplementation increased the capacity to produce ATP, with enriched mitochondrial coupling
efficiency identified as a key mechanism [120]. Similar to nitrate, nitrite treatment of rat aortic smooth
muscle cells increased mitochondrial quantity via enhanced mitochondrial biogenesis by altering the
expression and activity of AMP kinase and its downstream target, peroxisome proliferator-activated
receptor-γ coactivator 1α (PGC1α) [170]. Nitrite also protects mitochondria in models of hypoxia
[172] and after lethal doses of the inflammatory cytokine tumor necrosis factor-α (TNF-α) [173].
Collectively, these results offer compelling evidence that nitrate and nitrite supplementation may be
effective for normalizing and/or enhancing mitochondrial function in aging and disease.
Inflammation
Chronic low-grade inflammation increases with advancing age and contributes to several expressions
of physiological dysfunction [174, 175]. As an example, upregulation of pro-inflammatory cytokines,
such as IL-1β, IL-6, TNF-α, and interferon-γ, has been observed in the aorta of old mice when
compared to young controls. Sodium nitrite supplementation reversed levels of these inflammatory
cytokines to those seen in young animals, indicating a potent anti-inflammatory action [52]. The anti-
inflammatory effects of nitrite have also been shown in hypercholesterolemic mice, which experi-
enced an improvement in vascular function associated with a decline in C-reactive protein and
leukocyte markers of inflammation [162]. Additionally, nitrate and nitrite reduce inflammation in the
microvasculature. Pretreatment with nitrate and nitrite inhibited the migration of leukocytes after
myeloperoxidase-2 administration in the microvessels of mice. Furthermore, nitrate was shown to
have robust effects in reducing systemic inflammation in the presence of intestinal damage [176]. The
anti-inflammatory effects of nitrate have also been demonstrated. Four weeks of nitrate supplementa-
tion reduced age-associated increases in inflammatory macrophage migration inhibitory factor [79].
Moreover, nitrite decreases inflammation in models of endotoxemic shock [173, 177, 178], crush
injury [179], and ischemia–reperfusion injury [137–140, 180–182]. Taken together, these findings
support the possible use of nitrate and nitrite treatments for reducing inflammation with aging and
disease without compromising important aspects of immune function.
Metabolic Signaling
Recent evidence demonstrates that supplementation with sodium nitrite induces systemic changes in
multiple metabolic pathways in healthy older adults, as indicated by numerous alterations in the concen-
trations of small metabolites assessed via untargeted metabolomics analysis [55, 108]. Importantly,
many of these changes to the plasma metabolome in response to oral sodium nitrite are associated with
improvements of physiological function [55, 108]. Finally, baseline metabolic signatures can be used to
predict responsiveness (changes in physiological function) to a sodium nitrite intervention [55, 108].
Taken together, these new observations support the idea that nitrite/nitrate supplementation may produce
functional and health benefits through broad activation of metabolic signaling pathways.
Conclusions
Accumulating evidence suggests that nitrate and nitrite therapies hold promise for increasing NO
bioavailability independent of the endogenous
l
-arginine–NO-synthase pathway. In the setting of
aging, restoring NO bioavailability decreases oxidative stress via reduced free radical production and
L.C. Johnson et al.
271
increased antioxidant defenses, decreases inflammation, and normalizes mitochondrial function with
associated improvements in physiological outcomes. As the majority of work to date has been per-
formed in preclinical models or with acute dosing in humans, longer term studies with nitrate and
nitrite supplementation in humans are needed to establish efficacy of these strategies for preserving
physiological function and optimal health with advancing age.
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