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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
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 . 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
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
• 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.
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
-arginine–NO-synthase pathway in which a stimulus causes nitric oxide synthase (NOS) enzymes to
catalyze the reaction of
-arginine with oxygen to produce NO . 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) . 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.
NO . 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 .
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
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).
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 . 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
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 . As such, the endothelium plays an important role in maintaining vascu-
lar health .
Advancing age is associated with endothelial dysfunction, a pathological state of the endothelium
in which there is an imbalance between vasodilating and vasoconstricting factors . 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 . A primary hallmark of age-related endothelial dysfunction is the decreased synthesis
and/or bioavailability of NO .
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 , 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.
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  or 8 weeks of sodium nitrate  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 . 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 . 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 . 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 . Fragmentation of elastin and an increase in collagen
deposition contribute to reduced arterial compliance and stiffen the large elastic arteries . 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 . 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
, intra-arterial infusion of sodium nitrite , or dietary nitrate from beetroot juice  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 . 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 .
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  and
18 Nitrate and Nitrite in Aging and Age-Related Disease
older adults at risk for developing CVD . 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.
Older adults are at high risk for the development of hypertension , 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 , in clinical studies of healthy adults , in addition to patients with diabetes , pre-
hypertension , untreated hypertension , and urea cycle disorders . 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 . 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 . 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 . 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
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 . 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.
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 , 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 . In a subgroup analysis of patients treated with thrombolysis, nitrite also
reduced infarct size , 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 . 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 . Similarly, infusion of sodium nitrite markedly improved
forearm blood ﬂow in patients with congestive heart failure  and improved functional cardiac
responses during a dobutamine stress echocardiogram in patients with inducible myocardial ischemia
. 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 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 . 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 , which are consistent with general atrophy and loss
in brain volume . 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 ,
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
, and that oxidative stress and NO deﬁciency are key underlying factors [98, 99].
18 Nitrate and Nitrite in Aging and Age-Related Disease
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 . 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 . 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 .
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 . The effects of boosting NO
on learning and memory in rodents have been reviewed extensively elsewhere , 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 . 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 . 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 . 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.
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
. 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.
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 . 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 . 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 .
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 . 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
-arginine, a precursor of NO, improved mea-
sures of force production in postmenopausal women . 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 , 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 .
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 . 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 . 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
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 , and clinical investigations conﬁrm these effects in humans, with an acute
nitrite infusion reducing pulmonary pressures in states of hypoxia , and a single dose of dietary
nitrate improving exercise performance and blood pressure responses in patients with chronic obtru-
sive pulmonary disease .
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 , kidneys , liver , and lungs .
Furthermore, renal function is favorably affected by nitrite in other compromised states, such as renal
injury under conditions of eNOS inhibition  and brain death-mediated renal injury . Lastly,
nitrite is effective at improving function and reducing adverse outcomes in models of heart transplan-
tation . 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
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 . 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 . 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 , 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.
effects. Similarly, pentaerythritol tetranitrate, an organic nitrate, reduces NADPH oxidase activity in
the cardiac tissues of diabetic rats . 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.
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 . 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 . These investigations suggest nitrite supplementation may be an effec-
tive means to restore antioxidant defenses in aging and disease.
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
. Additionally, nitrite has been shown to improve EDD in hypercholesterolemic mice through main-
tenance of BH4/BH2 ratios . 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.
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 . 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
nitrate supplementation increased the capacity to produce ATP, with enriched mitochondrial coupling
efﬁciency identiﬁed as a key mechanism . 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) . Nitrite also protects mitochondria in models of hypoxia
 and after lethal doses of the inﬂammatory cytokine tumor necrosis factor-α (TNF-α) .
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.
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 . 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 . 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 . 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 .
Moreover, nitrite decreases inﬂammation in models of endotoxemic shock [173, 177, 178], crush
injury , 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.
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.
Accumulating evidence suggests that nitrate and nitrite therapies hold promise for increasing NO
bioavailability independent of the endogenous
-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.
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.
1. Niccoli T, Partridge L. Ageing as a risk factor for disease. Curr Biol. 2012;22(17):R741–52.
2. Heidenreich PA, Trogdon JG, Khavjou OA, Butler J, Dracup K, Ezekowitz MD, et al. Forecasting the future of cardio-
vascular disease in the United States: a policy statement from the American Heart Association. Circulation.
3. U.S. Census Bureau Population Division Table 12. Projections of the population by age and sex for the United States
4. Lunenfeld B, Stratton P. The clinical consequences of an ageing world and preventive strategies. Best Pract Res Clin
Obstet Gynaecol. 2013;27(5):643–59.
5. Kirkland JL. Translating advances from the basic biology of aging into clinical application. Exp Gerontol.
6. Seals DR, Justice JN, LaRocca TJ. Physiological geroscience: targeting function to increase healthspan and achieve
optimal longevity. J Physiol. 2016;594(8):2001–24.
7. Reutov VP, Sorokina EG. NO-synthase and nitrite-reductase components of nitric oxide cycle. Biochemistry.
8. Torregrossa AC, Aranke M, Bryan NS. Nitric oxide and geriatrics: implications in diagnostics and treatment of the
elderly. J Geriatr Cardiol. 2011;8(4):230–42.
9. Lundberg JO, Gladwin MT, Ahluwalia A, Benjamin N, Bryan NS, Butler A, et al. Nitrate and nitrite in biology, nutri-
tion and therapeutics. Nat Chem Biol. 2009;5(12):865–9.
10. Seals DR, Jablonski KL, Donato AJ. Aging and vascular endothelial function in humans. Clin Sci.
11. Kregel KC, Zhang HJ. An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and
pathological considerations. Am J Physiol Regul Integr Comp Physiol. 2007;292(1):R18–36.
12. Ferrer-Sueta G, Radi R. Chemical biology of peroxynitrite: kinetics, diffusion, and radicals. ACS Chem Biol.
13. Luo S, Lei H, Qin H, Xia Y. Molecular mechanisms of endothelial NO synthase uncoupling. Curr Pharm Des.
14. Bernard K, Hecker L, Luckhardt TR, Cheng G, Thannickal VJ. NADPH oxidases in lung health and disease. Antioxid
Redox Signal. 2014;20(17):2838–53.
15. Montezano AC, Touyz RM. Reactive oxygen species, vascular Noxs, and hypertension: focus on translational and
clinical research. Antioxid Redox Signal. 2014;20(1):164–82.
16. Lambeth JD. Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy. Free Radic Biol Med.
17. Dai DF, Rabinovitch PS, Ungvari Z. Mitochondria and cardiovascular aging. Circ Res. 2012;110(8):1109–24.
18. Schulz E, Wenzel P, Munzel T, Daiber A. Mitochondrial redox signaling: interaction of mitochondrial reactive oxygen
species with other sources of oxidative stress. Antioxid Redox Signal. 2014;20(2):308–24.
19. Chung JH, Seo AY, Chung SW, Kim MK, Leeuwenburgh C, Yu BP, et al. Molecular mechanism of PPAR in the regula-
tion of age-related inﬂammation. Ageing Res Rev. 2008;7(2):126–36.
20. Csiszar A, Wang M, Lakatta EG, Ungvari Z. Inﬂammation and endothelial dysfunction during aging: role of
NF-kappaB. J Appl Physiol. 2008;105(4):1333–41.
21. Sarkar D, Fisher PB. Molecular mechanisms of aging-associated inﬂammation. Cancer Lett. 2006;236(1):13–23.
22. Maggio M, Basaria S, Ble A, Lauretani F, Bandinelli S, Ceda GP, et al. Correlation between testosterone and the
inﬂammatory marker soluble interleukin-6 receptor in older men. J Clin Endocrinol Metab. 2006;91(1):345–7.
23. Bryan NS, Calvert JW, Gundewar S, Lefer DJ. Dietary nitrite restores NO homeostasis and is cardioprotective in
endothelial nitric oxide synthase-deﬁcient mice. Free Radic Biol Med. 2008;45(4):468–74.
24. Lundberg JO, Carlstrom M, Larsen FJ, Weitzberg E. Roles of dietary inorganic nitrate in cardiovascular health and
disease. Cardiovasc Res. 2011;89(3):525–32.
25. Rocha BS, Gago B, Pereira C, Barbosa RM, Bartesaghi S, Lundberg JO, et al. Dietary nitrite in nitric oxide biology: a
redox interplay with implications for pathophysiology and therapeutics. Curr Drug Targets. 2011;12(9):1351–63.
18 Nitrate and Nitrite in Aging and Age-Related Disease
26. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, et al. Heart disease and stroke statis-
tics—2015 update: a report from the American Heart Association. Circulation. 2015;131(4):e29–322.
27. Lakatta EG. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: part III: cellular and
molecular clues to heart and arterial aging. Circulation. 2003;107(3):490–7.
28. Cau SB, Carneiro FS, Tostes RC. Differential modulation of nitric oxide synthases in aging: therapeutic opportunities.
Front Physiol. 2012;3:218.
29. Najjar SS, Scuteri A, Lakatta EG. Arterial aging: is it an immutable cardiovascular risk factor? Hypertension.
30. Mitchell GF, Hwang SJ, Vasan RS, Larson MG, Pencina MJ, Hamburg NM, et al. Arterial stiffness and cardiovascular
events: the Framingham Heart Study. Circulation. 2010;121(4):505–11.
31. Mitchell GF. Effects of central arterial aging on the structure and function of the peripheral vasculature: implications
for end-organ damage. J Appl Physiol. 2008;105(5):1652–60.
32. Safar ME, Nilsson PM, Blacher J, Mimran A. Pulse pressure, arterial stiffness, and end-organ damage. Curr Hypertens
33. Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation. 2004;109(23 Suppl 1):III27–32.
34. Donato AJ, Morgan RG, Walker AE, Lesniewski LA. Cellular and molecular biology of aging endothelial cells. J Mol
Cell Cardiol. 2015;89(Pt B):122–35.
35. Luscher TF, Barton M. Biology of the endothelium. Clin Cardiol. 1997;20(11 Suppl 2):II-3–10.
36. Sindler AL, Devan AE, Fleenor BS, Seals DR. Inorganic nitrite supplementation for healthy arterial aging. J Appl
37. Lara J, Ashor AW, Oggioni C, Ahluwalia A, Mathers JC, Siervo M. Effects of inorganic nitrate and beetroot supple-
mentation on endothelial function: a systematic review and meta-analysis. Eur J Nutr. 2015;55(2):451–9.
38. Ignarro LJ, Gruetter CA. Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trini-
trate and sodium nitrite: possible involvement of S-nitrosothiols. Biochim Biophys Acta. 1980;631(2):221–31.
39. Gruetter CA, Gruetter DY, Lyon JE, Kadowitz PJ, Ignarro LJ. Relationship between cyclic guanosine 3′:5′-monophos-
phate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric
oxide: effects of methylene blue and methemoglobin. J Pharmacol Exp Ther. 1981;219(1):181–6.
40. Ignarro LJ, Lippton H, Edwards JC, Baricos WH, Hyman AL, Kadowitz PJ, et al. Mechanism of vascular smooth
muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of
S-nitrosothiols as active intermediates. J Pharmacol Exp Ther. 1981;218(3):739–49.
41. Moulds RF, Jauernig RA, Shaw J. A comparison of the effects of hydrallazine, diazoxide, sodium nitrite and sodium
nitroprusside on human isolated arteries and veins. Br J Clin Pharmacol. 1981;11(1):57–61.
42. Dejam A, Hunter CJ, Tremonti C, Pluta RM, Hon YY, Grimes G, et al. Nitrite infusion in humans and nonhuman
primates: endocrine effects, pharmacokinetics, and tolerance formation. Circulation. 2007;116(16):1821–31.
43. Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, et al. Nitrite reduction to nitric oxide by deoxyhe-
moglobin vasodilates the human circulation. Nat Med. 2003;9(12):1498–505.
44. Ingram TE, Pinder AG, Bailey DM, Fraser AG, James PE. Low-dose sodium nitrite vasodilates hypoxic human pul-
monary vasculature by a means that is not dependent on a simultaneous elevation in plasma nitrite. Am J Physiol Heart
Circ Physiol. 2010;298(2):H331–9.
45. Maher AR, Milsom AB, Gunaruwan P, Abozguia K, Ahmed I, Weaver RA, et al. Hypoxic modulation of exogenous
nitrite-induced vasodilation in humans. Circulation. 2008;117(5):670–7.
46. Mack AK, McGowan Ii VR, Tremonti CK, Ackah D, Barnett C, Machado RF, et al. Sodium nitrite promotes regional
blood ﬂow in patients with sickle cell disease: a phase I/II study. Br J Haematol. 2008;142(6):971–8.
47. Maher AR, Arif S, Madhani M, Abozguia K, Ahmed I, Fernandez BO, et al. Impact of chronic congestive heart failure
on pharmacokinetics and vasomotor effects of infused nitrite. Br J Pharmacol. 2013;169(3):659–70.
48. Heiss C, Meyer C, Totzeck M, Hendgen-Cotta UB, Heinen Y, Luedike P, et al. Dietary inorganic nitrate mobilizes
circulating angiogenic cells. Free Radic Biol Med. 2012;52(9):1767–72.
49. Ingram TE, Fraser AG, Bleasdale RA, Ellins EA, Margulescu AD, Halcox JP, et al. Low-dose sodium nitrite attenuates
myocardial ischemia and vascular ischemia-reperfusion injury in human models. J Am Coll Cardiol.
50. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, et al. Inorganic nitrate supplementation
lowers blood pressure in humans: role for nitrite-derived NO. Hypertension. 2010;56(2):274–81.
51. Joris PJ, Mensink RP. Beetroot juice improves in overweight and slightly obese men postprandial endothelial function
after consumption of a mixed meal. Atherosclerosis. 2013;231(1):78–83.
52. Sindler AL, Fleenor BS, Calvert JW, Marshall KD, Zigler ML, Lefer DJ, et al. Nitrite supplementation reverses vascu-
lar endothelial dysfunction and large elastic artery stiffness with aging. Aging Cell. 2011;10(3):429–37.
53. Rammos C, Totzeck M, Deenen R, Kohrer K, Kelm M, Rassaf T and Hendgen-Cotta UB. Dietary nitrate is a modiﬁer
of vascular gene expression in old male mice. Oxid Med Cell Longev 2015; 2015:658264 [Epub ahead of print].
54. Rammos C, Hendgen-Cotta UB, Sobierajski J, Bernard A, Kelm M, Rassaf T. Dietary nitrate reverses vascular dys-
function in older adults with moderately increased cardiovascular risk. Journal of the American College of Cardiology.
L.C. Johnson et al.
55. DeVan AE, Johnson LC, Brooks FA, Evans TD, Justice JN, Cruickshank-Quinn C, et al. Effects of sodium nitrite
supplementation on vascular function and related small metabolite signatures in middle-aged and older adults. J Appl
56. Zieman SJ, Melenovsky V, Kass DA. Mechanisms, pathophysiology, and therapy of arterial stiffness. Arterioscler
Thromb Vasc Biol. 2005;25(5):932–43.
57. Kliche K, Jeggle P, Pavenstadt H, Oberleithner H. Role of cellular mechanics in the function and life span of vascular
endothelium. Pﬂugers Arch. 2011;462(2):209–17.
58. Soldatos G, Cooper ME. Advanced glycation end products and vascular structure and function. Curr Hypertens Rep.
59. Katsuda S, Okada Y, Minamoto T, Oda Y, Matsui Y, Nakanishi I. Collagens in human atherosclerosis.
Immunohistochemical analysis using collagen type-speciﬁc antibodies. Arteriscler Thromb. 1992;12(4):494–502.
60. Semba RD, Sun K, Schwartz AV, Varadhan R, Harris TB, Satterﬁeld S, et al. Serum carboxymethyl-lysine, an advanced
glycation end product, is associated with arterial stiffness in older adults. J Hypertens. 2015;33(4):797–803;
61. Yoon SJ, Park S, Park C, Chang W, Cho DK, Ko YG, et al. Association of soluble receptor for advanced glycation
end-product with increasing central aortic stiffness in hypertensive patients. Coron Artery Dis. 2012;23(2):85–90.
62. Fleenor BS, Sindler AL, Eng JS, Nair DP, Dodson RB, Seals DR. Sodium nitrite de-stiffening of large elastic arteries
with aging: role of normalization of advanced glycation end-products. Exp Gerontol. 2012;47(8):588–94.
63. Bahra M, Kapil V, Pearl V, Ghosh S, Ahluwalia A. Inorganic nitrate ingestion improves vascular compliance but does
not alter ﬂow-mediated dilatation in healthy volunteers. Nitric Oxide. 2012;26(4):197–202.
64. Omar SA, Fok H, Tilgner KD, Nair A, Hunt J, Jiang B, 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):381–9, discussion 389.
65. Ghosh SM, Kapil V, Fuentes-Calvo I, Bubb KJ, Pearl V, Milsom AB, et al. Enhanced vasodilator activity of nitrite in
hypertension: critical role for erythrocytic xanthine oxidoreductase and translational potential. Hypertension.
66. Houston M, Hay J. Acute effects of an oral nitric oxide supplement on blood pressure, endothelial function, and vas-
cular compliance in hypertensive patients. J Clin Hypertens (Greenwich). 2014;16(7):524–9.
67. 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.
68. Rigaud A-S, Forette B. Hypertension in older adults. J Gerontol Ser A Biol Sci Med Sci. 2001;56(4):M217–25.
69. Montenegro MF, Pinheiro LC, Amaral JH, Marcal DM, Palei AC, Costa-Filho AJ, et al. Antihypertensive and antioxi-
dant effects of a single daily dose of sodium nitrite in a model of renovascular hypertension. Naunyn Schmiedebergs
Arch Pharmacol. 2012;385(5):509–17.
70. Pluta RM, Oldﬁeld EH, Bakhtian KD, Fathi AR, Smith RK, Devroom HL, et al. Safety and feasibility of long-term
intravenous sodium nitrite infusion in healthy volunteers. PLoS One. 2011;6(1), e14504.
71. Greenway FL, Predmore BL, Flanagan DR, Giordano T, Qiu Y, Brandon A, et al. Single-dose pharmacokinetics of
different oral sodium nitrite formulations in diabetes patients. Diabetes Technol Ther. 2012;14(7):552–60.
72. Biswas OS, Gonzalez VR, Schwarz ER. Effects of an oral nitric oxide supplement on functional capacity and blood
pressure in adults with prehypertension. J Cardiovasc Pharmacol Ther. 2015;20(1):52–8.
73. Nagamani SC, Campeau PM, Shchelochkov OA. Nitric-oxide supplementation for treatment of long-term complica-
tions in argininosuccinic aciduria. Am J Hum Genet. 2012;90(5):836–46.
74. Siervo M, Lara J, Jajja A, Sutyarjoko A, Ashor A, Brandt K, et al. Ageing modiﬁes the effects of beetroot juice supple-
mentation on 24-hour blood pressure variability: an individual participant meta-analysis. Nitric Oxide.
75. 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.
76. Bondonno CP, Liu AH, Croft KD, Ward NC, Shinde S, Moodley Y, et al. Absence of an effect of high nitrate intake
from beetroot juice on blood pressure in treated hypertensive individuals: a randomized controlled trial. Am J Clin
77. Siervo M, Lara J, Ogbonmwan I, Mathers JC. Inorganic nitrate and beetroot juice supplementation reduces blood pres-
sure in adults: a systematic review and meta-analysis. J Nutr. 2013;143(6):818–26.
78. Kelly J, Fulford J, Vanhatalo A, Blackwell JR, French O, Bailey SJ, et al. Effects of short-term dietary nitrate supple-
mentation on blood pressure, O2 uptake kinetics, and muscle and cognitive function in older adults. Am J Physiol
Regul Integr Comp Physiol. 2013;304(2):R73–83.
79. Rammos C, Hendgen-Cotta UB, Pohl J, Totzeck M, Luedike P, Schulze VT, et al. Modulation of circulating macro-
phage migration inhibitory factor in the elderly. Biomed Res Int. 2014;2014:582586.
80. Bryan NS, Calvert JW, Elrod JW, Gundewar S, Ji SY, Lefer DJ. Dietary nitrite supplementation protects against myo-
cardial ischemia-reperfusion injury. Proc Natl Acad Sci U S A. 2007;104(48):19144–9.
18 Nitrate and Nitrite in Aging and Age-Related Disease
81. Calvert JW, Lefer DJ. Myocardial protection by nitrite. Cardiovasc Res. 2009;83(2):195–203.
82. Webb A, Bond R, McLean P, Uppal R, Benjamin N, Ahluwalia A. Reduction of nitrite to nitric oxide during ischemia
protects against myocardial ischemia-reperfusion damage. Proc Natl Acad Sci U S A. 2004;101(37):13683–8.
83. Salloum FN, Sturz GR, Yin C, Rehman S, Hoke NN, Kukreja RC, et al. Beetroot juice reduces infarct size and
improves cardiac function following ischemia-reperfusion injury: possible involvement of endogenous H2S. Exp Biol
84. Siddiqi N, Neil C, Bruce M, MacLennan G, Cotton S, Papadopoulou S, et al. Intravenous sodium nitrite in acute
ST-elevation myocardial infarction: a randomized controlled trial (NIAMI). Eur Heart J. 2014;35(19):1255–62.
85. Jones DA, Pellaton C, Velmurugan S, Rathod KS, Andiapen M, Antoniou S, et al. Randomized phase 2 trial of intra-
coronary nitrite during acute myocardial infarction. Circ Res. 2015;116(3):437–47.
86. Paulus WJ, Tschope C. A novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myo-
cardial dysfunction and remodeling through coronary microvascular endothelial inﬂammation. J Am Coll Cardiol.
87. Zakeri R, Levine JA, Koepp GA, Borlaug BA, Chirinos JA, LeWinter M, et al. Nitrate’s effect on activity tolerance in
heart failure with preserved ejection fraction trial: rationale and design. Circ Heart Fail. 2015;8:221–8.
88. Zamani P, Rawat D, Shiva-Kumar P, Geraci S, Bhuva R, Konda P, et al. Effect of inorganic nitrate on exercise capacity
in heart failure with preserved ejection fraction. Circulation. 2015;131(4):371–80, discussion 380.
89. Buckner RL. Memory and executive function in aging and AD: multiple factors that cause decline and reserve factors
that compensate. Neuron. 2004;44(1):195–208.
90. Gunning-Dixon FM, Raz N. The cognitive correlates of white matter abnormalities in normal aging: a quantitative
review. Neuropsychology. 2000;14(2):224–32.
91. Fjell AM, Walhovd KB, Fennema-Notestine C, McEvoy LK, Hagler DJ, Holland D, et al. One-year brain atrophy
evident in healthy aging. J Neurosci. 2009;29(48):15223–31.
92. Ylikoski A, Erkinjuntti T, Raininko R, Sarna S, Sulkava R, Tilvis R. White matter hyperintensities on MRI in the
neurologically nondiseased elderly. Analysis of cohorts of consecutive subjects aged 55 to 85 years living at home.
93. Raz N. Aging of the brain and its impact on cognitive performance: integration of structural and functional ﬁndings.
In: Craik FIM, Salthouse TA, editors. Handbook of aging and cognition. 2nd ed. Mahwah: Erlbaum; 2000. p. 1–90.
94. Pantoni L, Garcia JH. Cognitive impairment and cellular/vascular changes in the cerebral white matter. Ann N Y Acad
95. Pugh KG, Lipsitz LA. The microvascular frontal-subcortical syndrome of aging. Neurobiol Aging. 2002;23(3):421–31.
96. de Groot JC, de Leeuw FE, Oudkerk M, van Gijn J, Hofman A, Jolles J, et al. Cerebral white matter lesions and cogni-
tive function: the Rotterdam Scan Study. Ann Neurol. 2000;47(2):145–51.
97. de Groot JC, De Leeuw FE, Oudkerk M, Van Gijn J, Hofman A, Jolles J, et al. Periventricular cerebral white matter
lesions predict rate of cognitive decline. Ann Neurol. 2002;52(3):335–41.
98. de la Torre JC, Stefano GB. Evidence that Alzheimer’s disease is a microvascular disorder: the role of constitutive
nitric oxide. Brain Res Brain Res Rev. 2000;34(3):119–36.
99. Floyd RA, Hensley K. Oxidative stress in brain aging. Implications for therapeutics of neurodegenerative diseases.
Neurobiol Aging. 2002;23(5):795–807.
100. Faraci FM. Protecting against vascular disease in brain. Am J Physiol Heart Circ Physiol. 2011;300(5):H1566–82.
101. Breteler MM, Bots ML, Ott A, Hofman A. Risk factors for vascular disease and dementia. Haemostasis.
102. Celsis P, Agniel A, Cardebat D, Demonet JF, Ousset PJ, Puel M. Age related cognitive decline: a clinical entity? A
longitudinal study of cerebral blood ﬂow and memory performance. J Neurol Neurosurg Psychiatry.
103. Girouard H, Iadecola C. Neurovascular coupling in the normal brain and in hypertension, stroke, and Alzheimer dis-
ease. J Appl Physiol. 2006;100(1):328–35.
104. Pitsikas N, Rigamonti AE, Cella SG, Sakellaridis N, Muller EE. The nitric oxide donor molsidomine antagonizes age-
related memory deﬁcits in the rat. Neurobiol Aging. 2005;26(2):259–64.
105. Paul V, Ekambaram P. Involvement of nitric oxide in learning & memory processes. Indian J Med Res. 2011;133(5):471.
106. Aamand R, Ho YC, Dalsgaard T, Roepstorff A, Lund TE. Dietary nitrate facilitates an acetazolamide-induced increase
in cerebral blood ﬂow during visual stimulation. J Appl Physiol. 2014;116(3):267–73.
107. Pluta RM, Dejam A, Grimes G, Gladwin MT, Oldﬁeld EH. Nitrite infusions to prevent delayed cerebral vasospasm in
a primate model of subarachnoid hemorrhage. JAMA. 2005;293(12):1477–84.
108. Justice JN, Johnson LC, DeVan AE, Cruickshank-Quinn C, Reisdorph N, Bassett CJ, et al. Improved motor and cogni-
tive performance with sodium nitrite supplementation is related to small metabolite signatures: a pilot trial in middle-
aged and older adults. Aging. 2015;7(11):1004–21.
109. Reid KF, Fielding RA. Skeletal muscle power: a critical determinant of physical functioning in older adults. Exerc
Sport Sci Rev. 2012;40(1):4.
L.C. Johnson et al.
110. Cooper R, Kuh D, Cooper C, Gale CR, Lawlor DA, Matthews F, et al. Objective measures of physical capability and
subsequent health: a systematic review. Age Ageing. 2011;40(1):14–23.
111. Studenski S, Perera S, Patel K, Rosano C, Faulkner K, Inzitari M, et al. Gait speed and survival in older adults. JAMA.
112. Fried LP, Guralnik JM. Disability in older adults: evidence regarding signiﬁcance, etiology, and risk. J Am Geriatr Soc.
113. Rantanen T, Guralnik JM, Sakari-Rantala R, Leveille S, Simonsick EM, Ling S, et al. Disability, physical activity, and
muscle strength in older women: the Women’s Health and Aging Study. Arch Phys Med Rehabil. 1999;80(2):130–5.
114. Doherty TJ. Invited review: aging and sarcopenia. J Appl Physiol. 2003;95(4):1717–27.
115. Vandervoort AA. Aging of the human neuromuscular system. Muscle Nerve. 2002;25(1):17–25.
116. Payne AM, Delbono O. Neurogenesis of excitation-contraction uncoupling in aging skeletal muscle. Exerc Sport Sci
117. Conley KE, Amara CE, Jubrias SA, Marcinek DJ. Mitochondrial function, ﬁbre types and ageing: new insights from
human muscle in vivo. Exp Physiol. 2007;92(2):333–9.
118. Verbrugge LM, Jette AM. The disablement process. Social Sci Med. 1994;38(1):1–14.
119. Hernández A, Schiffer TA, Ivarsson N, Cheng AJ, Bruton JD, Lundberg JO, et al. Dietary nitrate increases tetanic
[Ca2+] i and contractile force in mouse fast-twitch muscle. J Physiol. 2012;590(15):3575–83.
120. Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg JO, et al. Dietary inorganic nitrate improves
mitochondrial efﬁciency in humans. Cell Metab. 2011;13(2):149–59.
121. Heffernan KS, Chale A, Hau C, Cloutier GJ, Phillips EM, Warner P, et al. Systemic vascular function is associated with
muscular power in older adults. J Aging Res. 2012;2012:386387.
122. Ronnback M, Hernelahti M, Hamalainen E, Groop PH, Tikkanen H. Effect of physical activity and muscle morphol-
ogy on endothelial function and arterial stiffness. Scand J Med Sci Sports. 2007;17(5):573–9.
123. Justice JN, Gioscia-Ryan RA, Johnson LC, Battson ML, de Picciotto NE, Beck HJ, et al. Sodium nitrite supplementa-
tion improves motor function and skeletal muscle inﬂammatory proﬁle in old male mice. J Appl Physiol (1985).
124. Cermak NM, Gibala MJ, van Loon LJ. Nitrate supplementation’s improvement of 10-km time-trial performance in
trained cyclists. Int J Sport Nutr Exerc Metab. 2012;22(1):64–71.
125. Lansley KE, Winyard PG, Fulford J, Vanhatalo A, Bailey SJ, Blackwell JR, et al. Dietary nitrate supplementation
reduces the O2 cost of walking and running: a placebo-controlled study. J Appl Physiol. 2011;110(3):591–600.
126. Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Effects of dietary nitrate on oxygen cost during exercise. Acta
Physiol (Oxf). 2007;191(1):59–66.
127. Fricke O, Baecker N, Heer M, Tutlewski B, Schoenau E. The effect of l‐arginine administration on muscle force and
power in postmenopausal women. Clin Physiol Funct Imaging. 2008;28(5):307–11.
128. Casey DP, Treichler DP, Ganger CT, Schneider AC, Ueda K. Acute dietary nitrate supplementation enhances compen-
satory vasodilation during hypoxic exercise in older adults. J Appl Physiol. 2015;118(2):178–86.
129. Kenjale AA, Ham KL, Stabler T, Robbins JL, Johnson JL, Vanbruggen M, et al. Dietary nitrate supplementation
enhances exercise performance in peripheral arterial disease. J Appl Physiol. 2011;110(6):1582–91.
130. Baliga RS, Milsom AB, Ghosh SM, Trinder SL, Macallister RJ, Ahluwalia A, et al. Dietary nitrate ameliorates pulmo-
nary hypertension: cytoprotective role for endothelial nitric oxide synthase and xanthine oxidoreductase. Circulation.
131. Berry MJ, Justus NW, Hauser JI, Case AH, Helms CC, Basu S, et al. Dietary nitrate supplementation improves exer-
cise performance and decreases blood pressure in COPD patients. Nitric Oxide. 2014;48:22–30.
132. Dezfulian C, Alekseyenko A, Dave KR, Raval AP, Do R, Kim F, et al. Nitrite therapy is neuroprotective and safe in
cardiac arrest survivors. Nitric Oxide. 2012;26(4):241–50.
133. Shiva S, Sack MN, Greer JJ, Duranski M, Ringwood LA, Burwell L, et al. Nitrite augments tolerance to ischemia/
reperfusion injury via the modulation of mitochondrial electron transfer. J Exp Med. 2007;204(9):2089–102.
134. Wang WZ, Fang XH, Stephenson LL, Zhang X, Williams SJ, Baynosa RC, et al. Nitrite attenuates ischemia-reperfu-
sion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle.
Plast Reconstr Surg. 2011;128(4):279e–87.
135. Tripatara P, Patel NS, Webb A, Rathod K, Lecomte FM, Mazzon E, et al. Nitrite-derived nitric oxide protects the rat
kidney against ischemia/reperfusion injury in vivo: role for xanthine oxidoreductase. J Am Soc Nephrol.
136. Duranski MR, Greer JJ, Dejam A, Jaganmohan S, Hogg N, Langston W, et al. Cytoprotective effects of nitrite during
in vivo ischemia-reperfusion of the heart and liver. J Clin Invest. 2005;115(5):1232–40.
137. Sugimoto R, Okamoto T, Nakao A, Zhan J, Wang Y, Kohmoto J, et al. Nitrite reduces acute lung injury and improves
survival in a rat lung transplantation model. Am J Transplant. 2012;12(11):2938–48.
138. Milsom AB, Patel NS, Mazzon E, Tripatara P, Storey A, Mota-Filipe H, et al. Role for endothelial nitric oxide synthase
in nitrite-induced protection against renal ischemia-reperfusion injury in mice. Nitric Oxide. 2010;22(2):141–8.
18 Nitrate and Nitrite in Aging and Age-Related Disease
139. Kelpke SS, Chen B, Bradley KM, Teng X, Chumley P, Brandon A, et al. Sodium nitrite protects against kidney injury
induced by brain death and improves post-transplant function. Kidney Int. 2012;82(3):304–13.
140. Zhan J, Nakao A, Sugimoto R, Dhupar R, Wang Y, Wang Z, et al. Orally administered nitrite attenuates cardiac
allograft rejection in rats. Surgery. 2009;146(2):155–65.
141. Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol.
142. Venkataraman K, Khurana S, Tai TC. Oxidative stress in aging—matters of the heart and mind. Int J Mol Sci.
143. Romano AD, Serviddio G, de Matthaeis A, Bellanti F, Vendemiale G. Oxidative stress and aging. J Nephrol. 2010;23
144. Bachschmid MM, Schildknecht S, Matsui R, Zee R, Haeussler D, Cohen RA, et al. Vascular aging: chronic oxidative
stress and impairment of redox signaling-consequences for vascular homeostasis and disease. Ann Med.
145. Paneni F, Costantino S, Cosentino F. Molecular pathways of arterial aging. Clin Sci (Lond). 2015;128(2):69–79.
146. Ungvari Z, Kaley G, de Cabo R, Sonntag WE, Csiszar A. Mechanisms of vascular aging: new perspectives. J Gerontol
A Biol Sci Med Sci. 2010;65(10):1028–41.
147. Zhu SG, Kukreja RC, Das A, Chen Q, Lesnefsky EJ, Xi L. Dietary nitrate supplementation protects against Doxorubicin-
induced cardiomyopathy by improving mitochondrial function. J Am Coll Cardiol. 2011;57(21):2181–9.
148. Carlstrom M, Persson AE, Larsson E, Hezel M, Scheffer PG, Teerlink T, et al. Dietary nitrate attenuates oxidative
stress, prevents cardiac and renal injuries, and reduces blood pressure in salt-induced hypertension. Cardiovasc Res.
149. Bir SC, Pattillo CB, Pardue S, Kolluru GK, Docherty J, Goyette D, et al. Nitrite anion stimulates ischemic arteriogen-
esis involving NO metabolism. Am J Physiol Heart Circ Physiol. 2012;303(2):H178–88.
150. Oudot A, Martin C, Busseuil D, Vergely C, Demaison L, Rochette L. NADPH oxidases are in part responsible for
increased cardiovascular superoxide production during aging. Free Radic Biol Med. 2006;40(12):2214–22.
151. Wang M, Zhang J, Walker SJ, Dworakowski R, Lakatta EG, Shah AM. Involvement of NADPH oxidase in age-asso-
ciated cardiac remodeling. J Mol Cell Cardiol. 2010;48(4):765–72.
152. Krause K-H. Aging: a revisited theory based on free radicals generated by NOX family NADPH oxidases. Exp
153. Schuhmacher S, Oelze M, Bollmann F, Kleinert H, Otto C, Heeren T, et al. Vascular dysfunction in experimental
diabetes is improved by pentaerithrityl tetranitrate but not isosorbide-5-mononitrate therapy. Diabetes.
154. İnal ME, Kanbak G, Sunal E. Antioxidant enzyme activities and malondialdehyde levels related to aging. Clin Chim
155. Corbi G, Conti V, Russomanno G, Rengo G, Vitulli P, Ciccarelli AL, et al. Is physical activity able to modify oxidative
damage in cardiovascular aging? Oxid Med Cell Longev. 2012;2012:728547.
156. Singh M, Arya A, Kumar R, Bhargava K, Sethy NK. Dietary nitrite attenuates oxidative stress and activates antioxidant
genes in rat heart during hypobaric hypoxia. Nitric oxide : biology and chemistry/ofﬁcial journal of the Nitric Oxide
157. Lu XX, Wang SQ, Zhang Z, Xu HR, Liu B, Huangfu CS. [Protective effects of sodium nitrite preconditioning against
alcohol-induced acute liver injury in mice]. Sheng Li Xue Bao [Acta Physiologica Sinica]. 2012;64(3):313–20.
158. Doganci S, Yildirim V, Bolcal C, Korkusuz P, Gumusel B, Demirkilic U, et al. Sodium nitrite and cardioprotective
effect in pig regional myocardial ischemia-reperfusion injury model. Adv Clin Exp Med. 2012;21(6):713–26.
159. Perlman DH, Bauer SM, Ashraﬁan H, Bryan NS, Garcia-Saura MF, Lim CC, et al. Mechanistic insights into nitrite-
induced cardioprotection using an integrated metabolomic/proteomic approach. Circ Res. 2009;104(6):796–804.
160. Mohler III ER, Hiatt WR, Gornik HL, Kevil CG, Quyyumi A, Haynes WG, et al. Sodium nitrite in patients with
peripheral artery disease and diabetes mellitus: safety, walking distance and endothelial function. Vasc Med.
161. Delp MD, Behnke BJ, Spier SA, Wu G, Muller-Delp JM. Ageing diminishes endothelium-dependent vasodilatation
and tetrahydrobiopterin content in rat skeletal muscle arterioles. J Physiol. 2008;586(4):1161–8.
162. Stokes KY, Dugas TR, Tang Y, Garg H, Guidry E, Bryan NS. Dietary nitrite prevents hypercholesterolemic microvas-
cular inﬂammation and reverses endothelial dysfunction. Am J Physiol Heart Circ Physiol. 2009;296(5):H1281–8.
163. Ungvari Z, Sonntag WE, Csiszar A. Mitochondria and aging in the vascular system. J Mol Med (Berl).
164. Kluge MA, Fetterman JL, Vita JA. Mitochondria and endothelial function. Circ Res. 2013;112(8):1171–88.
165. Shenouda SM, Widlansky ME, Chen K, Xu G, Holbrook M, Tabit CE, et al. Altered mitochondrial dynamics contrib-
utes to endothelial dysfunction in diabetes mellitus. Circulation. 2011;124(4):444–53.
166. Gioscia-Ryan RA, LaRocca TJ, Sindler AL, Zigler MC, Murphy MP, Seals DR. Mitochondria-targeted antioxidant
(MitoQ) ameliorates age-related arterial endothelial dysfunction in mice. J Physiol. 2014;592(Pt 12):2549–61.
L.C. Johnson et al.
167. Miller MW, Knaub LA, Olivera-Fragoso LF, Keller AC, Balasubramaniam V, Watson PA, et al. Nitric oxide regulates
vascular adaptive mitochondrial dynamics. Am J Physiol Heart Circ Physiol. 2013;304(12):H1624–33.
168. Shiva S. Nitrite: A physiological store of nitric oxide and modulator of mitochondrial function. Redox Biol.
169. Shiva S, Rassaf T, Patel RP, Gladwin MT. The detection of the nitrite reductase and NO-generating properties of hae-
moglobin by mitochondrial inhibition. Cardiovasc Res. 2011;89(3):566–73.
170. Mo L, Wang Y, Geary L, Corey C, Alef MJ, Beer-Stolz D, et al. Nitrite activates AMP kinase to stimulate mitochon-
drial biogenesis independent of soluble guanylate cyclase. Free Radic Biol Med. 2012;53(7):1440–50.
171. Murillo D, Kamga C, Mo L, Shiva S. Nitrite as a mediator of ischemic preconditioning and cytoprotection. Nitric
172. Kamga Pride C, Mo L, Quesnelle K, Dagda RK, Murillo D, Geary L, et al. Nitrite activates protein kinase A in nor-
moxia to mediate mitochondrial fusion and tolerance to ischaemia/reperfusion. Cardiovasc Res. 2014;101(1):57–68.
173. Cauwels A, Buys ES, Thoonen R, Geary L, Delanghe J, Shiva S, et al. Nitrite protects against morbidity and mortality
associated with TNF- or LPS-induced shock in a soluble guanylate cyclase-dependent manner. J Exp Med.
174. Michaud M, Balardy L, Moulis G, Gaudin C, Peyrot C, Vellas B, et al. Proinﬂammatory cytokines, aging, and age-
related diseases. J Am Med Dir Assoc. 2013;14(12):877–82.
175. Wang M, Jiang L, Monticone RE, Lakatta EG. Proinﬂammation: the key to arterial aging. Trends Endocrinol Metab.
176. Jadert C, Petersson J, Massena S, Ahl D, Grapensparr L, Holm L, et al. Decreased leukocyte recruitment by inorganic
nitrate and nitrite in microvascular inﬂammation and NSAID-induced intestinal injury. Free Radic Biol Med.
177. Cauwels A, Brouckaert P. Nitrite regulation of shock. Cardiovasc Res. 2011;89(3):553–9.
178. Hamburger T, Broecker-Preuss M, Hartmann M, Schade FU, de Groot H, Petrat F. Effects of glycine, pyruvate, resve-
ratrol, and nitrite on tissue injury and cytokine response in endotoxemic rats. J Surg Res. 2013;183(1):e7–21.
179. Murata I, Nozaki R, Ooi K, Ohtake K, Kimura S, Ueda H, et al. Nitrite reduces ischemia/reperfusion-induced muscle
damage and improves survival rates in rat crush injury model. J Trauma Acute Care Surg. 2012;72(6):1548–54.
180. Okamoto T, Tang X, Janocha A, Farver CF, Gladwin MT, McCurry KR. Nebulized nitrite protects rat lung grafts from
ischemia reperfusion injury. J Thorac Cardiovasc Surg. 2013;145(4):1108–16.
181. Pattillo CB, Fang K, Terracciano J, Kevil CG. Reperfusion of chronic tissue ischemia: nitrite and dipyridamole regula-
tion of innate immune responses. Ann N Y Acad Sci. 2011;1207:83–8.
182. Pattillo CB, Fang K, Pardue S, Kevil CG. Genome expression proﬁling and network analysis of nitrite therapy during
chronic ischemia: possible mechanisms and interesting molecules. Nitric Oxide. 2011;22(2):168–79.
18 Nitrate and Nitrite in Aging and Age-Related Disease