IntroductionThe past 100 years have seen many scientific and medical discoveries leading to tremendo...

1. A holistic approach to modern-day chronic disease
management: pharmacological therapies, lifestyle choices,
and nitric oxide deficiency.
Introduction
The past 100 years have seen many scientific and medical discoveries leading to tremendous
advancements in medicine worldwide. Antibiotics/antiseptics and vaccinations have led to a drastic
reduction in the morbidity burden of infectious diseases. The advent of medical devices and
advancements in emergency medicine have led to better care of trauma patients and life-threatening
emergencies from acute injury. Innovations in imaging and diagnostics have led to early detection of
a number of chronic diseases. However, the development of safe and effective treatments or cures of
chronic diseases such as cardiovascular disease, Alzheimer's disease, cancer, and diabetes have
been disappointingly slow and largely ineffective. According to the 2010 National Center for Health
Statistics Report, life expectancy has increased 1.1 years over the past decade, going from 76.8 to
77.9. (1) All causes of death adjusted for age have decreased by 12.5% from 2000 to 2008. However,
the percent of the population 18 years and over with heart disease has risen from 10.9% to 11.8%
and the population 65 and over has risen from 29.6% to 31.7% over the same 8 years. Diabetes has
gone from 8.5% of the population 20 years and older to 11.9% in just 8 years. The percentage of
people with hypertension has risen from 28.9% to 32.6%. Cancer has followed a similar trend,
increasing from 4.9% to 6.1% in patients 18 years old and over. (1) Essentially, although the life
expectancy of the average population has increased, the population is living better or healthier. A
number of the increased years of life are spent with a chronic disease condition, which requires
intensive treatment and care. This causes an enormous economic burden on the health-care system
and, consequently, on the patients. In fact from 2000 to 2008, total health-care expenditures
increased from $1.1 to $2.0 trillion dollars or from $4032 to $6411 per capita. (1) These data
highlight a very serious problem with our health-care system.
While it is highly unlikely that a single event or mediator may be responsible for this trend in health
and in complex chronic diseases, there is emerging evidence that nitric oxide (NO) may play a role at
the earliest stages of chronic diseases. Modern pharmacotherapy and lifestyle choices are
increasingly being linked to diminished NO levels in vivo, and therefore NO levels should be a
primary consideration when considering the health of an individual. The discovery of NO in the
1980s as a vasodilator and signaling molecule in the cardiovascular system, immune system, and
nervous system marked a point of inflexion in medicine. (2-5) The discovery that a simple molecule
produced as a gas could perform so many essential and critical biological and physiological functions
established a new paradigm in cell signaling. Now, almost 30 years later, endothelial dysfunction or
insufficient NO production is recognized as the earliest event in the onset and progression of a
number of chronic diseases. Loss of endothelial NO function is associated with several
cardiovascular disorders, including atherosclerosis, which is due either to decreased production or
to increased degradation of NO. (6) A number of studies provide evidence that endothelial NO
dysfunction is not only associated with all major cardiovascular risk factors, such as hyperlipidemia,
diabetes, hypertension, smoking, and severity of atherosclerosis, but also has a profound predictive
value for future atherosclerotic disease progression. (7-10) There is also an increasing evidence pool
linking NO to neurodegenerative diseases, such Alzheimer's disease (AD). Decreased levels of nitrite
and nitrate (collectively referred to as NOx) have been detected in patients with different forms of

2. dementia, especially AD. (11) The exact etiology of sporadic AD is unclear, but it is interesting that
cardiovascular risk factors including hypertension, hypercholesterolemia, diabetes mellitus, aging,
and sedentary lifestyle are associated with higher incidence of AD (all conditions associated with NO
insufficiency). (12) The link between cardiovascular risk factors and AD has yet to be identified;
however, a common feature is endothelial dysfunction; specifically, decreased bioavailability of NO.
(13)
Insulin has vasodilator actions that depend on endothelium-derived NO. (14) Type 2 diabetes
mellitus accounts for 80% to 90% of diabetes cases in the US and is associated with an increased
risk for a number of life-threatening complications. These include heart disease and stroke, high
blood pressure, blindness, kidney disease, nervous system disease, amputation, and complications of
pregnancy and surgery. Incidentally, all of the above complications are associated with insufficient
NO production. (15) Endothelial dysfunction with reduced NO generation and bioavailability plays a
key role in the pathogenesis of diabetic vascular disease and complications and likely serves as the
key link between metabolic disorders and cardiovascular disease (CVD). (16)
Medical literature is rich with clear assocaitions and near causal relationships between NO levels
and onset and progression of chronic diseases, yet there are no protocols to use NO in the
treatment/therapautic domain. Some common prescription medications even reduced NO
bioavalibitiy and production. Many lifestyle and dietary habits that lead to chronic disease are linked
to insufficient NO production or availability and the standard of care for patients in intensive care
units. An overview of select lifestyle choices and medical procedures and pharmacological therapies
that are known to be linked to NO will be reviewed.
Diet and Nutrition
There is now an established and accepted role for nitrite and nitrate in the diet contributing to NO
homeostasis. (17-19) In fact, the nitrate-nitrite-nitric oxide pathway has been suggested and
demonstrated to be the mechanism of action for health benefits from certain diets. (20), (21) The
Dietary Approaches to Stop Hypertension (DASH) studies found that diets rich in vegetables (i.e., 8-
10 servings) and low-fat dairy products could lower blood pressure to an extent similar to single
hypotensive medications.n" These and other findings point to a widely acknowledged hypothesis that
the content of inorganic nitrate (NO3--) in certain vegetables provides a physiologic substrate for
reduction to nitrite (NO2 --), NO, and other metabolic products that produce vasodilation, decrease
blood pressure, and support cardiovascular function. (20), (24-26) It has been reported that dietary
nitrate reduces blood pressure in healthy volunteers, prevents platelet aggregation, and is
vasoprotective. (27-29) One of early studies indicates that homeostatic levels of tissue nitrite and
nitrate are affected by dietary NOx intake. (30) Furthermore, enriching dietary intake of nitrite and
nitrate in mice translates into significantly less injury from heart attack, and diets with insufficient
nitrite and nitrate cause increased injury and increased mortality. (31) Nitrite in the diet can prevent
microvascular inflammation and restore normal endothelial function. (32) Dietary nitrate has also
been demonstrated to reduce leukocyte recruitment to inflammation in a process involving
attenuation of P-selectin and ICAM-1 upregulation.n Nitrite supplementation reverses vascular
endothelial dysfunction and large elastic artery stiffness with aging. (34) Emerging evidence now
suggests antioxidant effects of nitrate/nitrite. (32), (35-37) Dietary nitrate has been shown to reverse
metabolic syndrome in experimental animals. (38)
Furthermore, in the stomach, nitrite-derived NO seems to play an important role in host defense and
in regulation of gastric mucosal integrity. (39), (40) Oral nitrite has also been shown to reverse L-
NAME induced hypertension and serve as an alternate source of NO in vivo. (41) Nitrite and nitrate
therapy or supplementation may restore NO homeostasis from endothelial dysfunction and provide

3. benefit in a number of diseases characterized by NO insufficiency. (18) This will provide a host of
new dietary guidelines for maintaining good health and a solid basis for new preventative and
therapeutic regimens. However, this requires sufficient ingestion from eating nitrate-enriched foods.
Unfortunately for those in the US, the typical Western diet is composed of dairy products, cereals,
refined cereals, refined sugars, refined vegetable oils, fatty meats, salt, and combinations of these
foods, which are not good sources of nitrite and nitrate. (26), (42) Fresh, leafy vegetables are
especially rich in nitrite and nitrate, and incorporating these into the daily Western diet would thus
lead to better disease prevention. Long-term trials and epidemiological research will be needed to
confirm this hypothesis.
Antiseptic Mouthwash/Antibiotics
The bioactivation of nitrate from dietary or endogenous sources requires its initial reduction to
nitrite, and because mammals lack specific and effective nitrate reductase enzymes, this conversion
is mainly carried out by commensal bacteria in the mouth and gastrointestinal tract and on body
surfaces. (39) Dietary nitrate is rapidly absorbed in the upper gastrointestinal tract. In the blood, it
mixes with the nitrate formed from the oxidation of endogenous NO produced from the nitric oxide
synthase (NOS) enzymes. After a meal rich in nitrate, the levels in plasma increase and remain high
for a prolonged period of time (plasma half-life of nitrate is 5-6 hours). The nitrite levels in plasma
also increase after nitrate ingestion in some persons. (43) Nitrate (approximately 25%) is actively
taken up by the salivary glands and is concentrated up to 20-fold in saliva. (43), (44) Once in the
mouth, commensal facultative anaerobic bacteria reduce nitrate to nitrite during respiration by the
action of nitrate reductases. (39), (45) The salivary nitrate levels can approach 10 mM and nitrite
levels 1 to 2 mM after a dietary nitrate load. (43) When saliva enters the acidic stomach (1-1.5 L per
day), much of the nitrite is rapidly protonated to form nitrous acid (HNO2; pKa 3.3), which
decomposes further to form NO and other nitrogen oxides. (46), (47) The enterosalivary circulation
of nitrate and subsequent reduction to nitrite in saliva can equate to as much as 40 to 100 mg of
nitrite per day from eating a high-nitrate meal. This dietary pathway for generation of NO is
dependent upon select bacteria in the mouth and sufficient acid production in the stomach. Research
groups at the Karolinska Institute have convincingly shown that use of antiseptic mouthwash or
abolishing gastrointestinal bacteria affect endogenous NO production. (48-50) Patients on broad-
spectrum antibiotics are at increased risk of opportunistic gastrointestinal tract infections from
species such as Clostridium alhicans and Clostridium difficile. The overuse of antibiotics might be
responsible for killing off the commensal bacteria responsible for salivary nitrate reduction, and thus
reducing salivary nitrite that allows for the same bacteria to be established in the Cl tract.
The salivary reduction mechanism might also play a role in the innate immune response and wound
healing. Both humans and animals will lick their wounds upon sustaining injury, an action that
promotes wound healing. (51) Although numerous innate immune defense mechanisms and
immunoglobulins are present in saliva, part of the mechanism may in fact be through an NO
pathway by salivary nitrite. (52) Licking of human skin results in production of NO as salivary nitrite
is reduced on the acidic skin surface. (53) A number of common skin pathogens are effectively killed
by acidified nitrite. (54) The acidification of nitrite from dietary nitrate likely plays a role in the
wound healing aspect of saliva.
Processes that kill or disrupt commensal bacterial function in the mouth, GI tract, and skin are likely
to disrupt NO production. Everyday actions such as the use of antibacterial soaps, antiseptic
mouthwash, overuse of antibiotics, and even seemingly beneficial activities, such as bathing twice a
day, affect fundamental pathways for NO production. It is critical to remember that not all bacteria
are bad and that the numbers of commensal bacteria that reside in our body outnumber our own
human cells 10 to 1. The majority of the commensal bacterial in the human body play incredibly vital

4. roles, and maintaining communities of these while combating infectious or pathogenic bacteria
should be a balanced effort and the goal of hygiene and antibiotic use.
Proton Pump Inhibitors
Stomach acid levels directly correlate to the nitrate reduction cycle in the mouth. Nitrite produced
on the dorsal surface of the tongue by bacterial reduction is further reduced to NO under acidic
conditions and result in the destruction of acid-producing organisms. (39) NO from acidic reduction
of nitrite, along with other reactive nitrogen species, is produced in the stomach following a nitrate-
rich meal in quantities far greater than that required to produce vasodilatation, suggesting a role
other than modulation of gastric blood flow. (55) A number of important human pathogens (C.
albicans, E. coli 0157, Salmonella, Shigella) are resistant to a low pH found in the stomach, which
enables them to survive passage through the stomach. (56) The addition of nitrite in concentrations
found in human saliva results in much more effective killing of these pathogens than in acid alone.
(47), (57) Indeed even H. pylori, a bacterium well adapted to colonize the human stomach, is
susceptible to acidified nitrite. (58) Acidified nitrite plays a pivotal role in protection against
ingested pathogens. Further support for this can be found in the reduced levels of postprandial
salivary nitrite found in subjects treated with the broad-spectrum antibiotic amoxicillin. (59) Since
the pKa of nitrite is approximately 3.3, in order to effectively generate NO disproportionation, the
stomach must be acidic. A reduction of acid production, and the resulting pH spike, leads to a
disruption of this pathway. Achlorhydria has been defined by multiple separate systems in reference
to gastric acid secretion. First, achlorhydria has been defined by a peak acid output in response to a
maximally effective stimulus that results in an intragastric pH of greater than 5.09 in men and
greater than 6.81 in women. Second, achlorhydria has been defined by a maximal acid output of less
than 6.9 m/moleih in men and less than 5.0 m/mole/h in women. Third, achlorhydria has been
defined as a ratio of serum pepsinogen I/pepsinogen II of less than 2.9. It is estimated that
approximately 30% of the population over 65 years are hypochlorhydric. Several medical conditions
and specific gastric surgery can lead to achlorhydria. Several conditions associated with
achlorhydria lead to increased mortality and morbidity. Specifically, achlorhydria has been
associated with gastric cancer, hip fracture, and bacterial overgrowth, all of which may be partly
due to disruption of NO production from nitrite.
Proton pump inhibitors (PPIs) are the third-highest selling class of drugs in the US, with $13.9
billion in annual sales and a staggering 113.4 million prescriptions filled each year. The drugs are
used for gastroesophogeal reflux disease (GERD) and bleeding peptic ulcers. Yet a study found that
high-dose PPIs, when compared with low-dose PPIs, do not further reduce the rates of rebleeding,
need for surgical intervention, or mortality after endoscopic treatment in patients with bleeding
peptic ulcers. (60) There is emerging evidence that chronic use of PPIs is associated with a number
of other health problems that appear to be related to NO biology. Postmenopausal women who used
PPIs had a 26% increased risk for forearm and wrist fractures, a 47% increased risk for spine
fractures, and a 25% increased risk for total fractures. (61) Long-term PPI therapy, particularly at
high doses, is associated with an increased risk of hip fracture. (62) The mortality rate during the
first year after a hip fracture is 20%. Among those who survive, 1 in 5 patients require nursing home
care. Several potential mechanisms may explain this association. Significant hypochlorhydria,
particularly among the elderly, who may have a higher prevalence of H. pylori infection, could result
in calcium malabsorption secondary to small bowel bacterial overgrowth. Limited animal and human
studies have shown that PPI therapy may decrease insoluble calcium absorption or bone density.
(63) NO, in addition to its known cardioprotective effects, appears to also play a role in osteoblast
function and bone turnover. (64) Supporting this notion, in a small randomized controlled trial,
nitroglycerine (an NO donor) was found to be as effective as estrogen in preventing bone loss in
women with surgical menopause. (65) Disruption of the acid disproportionation of nitrite to nitric

5. oxide through PPI use may also affect the NO biology of bone metabolism.
Achlorhydria is an important cause of hypergastrinemia, which can subsequently lead to the
development of GI carcinoid tumors. There are now enough data from patients who have been taking
PPIs for sufficient periods to begin to assess the consequences of long term therapy and it appears
that chronic use of these is associated with gastric carcinoid formation. (66) It is not clear if the
reduction in NO production in the stomach is causal for increased incidence of carcinoid tumors. The
safety of long-term PPI administration needs serious prospective study. I would strongly suggest that
the role of NO be investigated in such studies.
NSAIDS
Prostacyclin (PGI2) is a vasodilator and an inhibitor of platelet aggregation, properties consistent
with cardioprotection. More than a decade ago, inhibition of cyclooxygenase-2 (COX-2) by the
nonsteroidal anti-inflammatory drugs (NSAIDs) rofecoxib (Vioxx) and celecoxib (Celebrex) was found
to reduce the amount of the major metabolite of PGI2 in the urine of healthy volunteers. This
suggested that this particular class of NSAIDs might cause adverse cardiovascular events by
reducing production of cardioprotective PGI2. This prediction was based on the assumption that the
concentration of PGI in urine likely reflected vascular production of PGI2 and that other
cardioprotective mediators, especially NO, were not able to compensate for the loss of PGI2. A
number of placebo-controlled clinical trials showed that NSAIDs that block COX-2 increase adverse
cardiovascular events. (67) In fact, the reason that drugs such as Vioxx and Celebrex were recalled
by the FDA was because of concerns of these side effects. Recent basic science data reveal that
deletion of COX-2 enzyme in the mouse vasculature reduces excretion of PGI2 in urine and
predisposes the animals to both hypertension and thrombosis. Furthermore, vascular disruption of
COX-2 depressed expression of endothelial NO synthase and the consequent release and function of
NO.68 Thus, suppression of PGI2 formation resulting from deletion of vascular COX-2 is sufficient to
explain the cardiovascular hazard from NSAIDs, which is likely to be augmented by secondary
mechanisms such as suppression of NO production. Recognition of a role of NO during COX-2
inhibition opened the door for novel NO-hybrid drugs such as NO-naproxen or NO hybrids of
selective COX-2 inhibitors. Conventional reasoning was that if you could provide a source of NO
along with the parent drug, one could overcome the cardiovascular complications from such
pharmaceuticals. Unfortunately, this strategy has not proved safe and effective as the lead
compound, NO-naproxen (naproxcinod), was not approved by the FDA. Early consideration and/or
recognition of the effects of COX-2 inhibitors on endogenous NO production may have delayed its
entry into the market and prevented many of the adverse events from these drugs. As such all new
and existing classes of pharmacological therapies should consider and assess effect on NO
production/activity.
Other nonspecific NSAIDs such as aspirin, naproxen, and cliclofenac are highly effective drugs that
inhibit pain and inflammation; and perhaps due to the role of inflammation in the underlying
etiology, the utility of these powerful drugs is limited due to their gastrointestinal (GI) side effects,
notably peptic ulceration and GI bleeding. (69) It has been demonstrated that pretreatment with
dietary nitrate protects against diclofenacs induced gastric ulcers likely via enhanced nitrite-
dependent intragastric NO formation and concomitant stimulation of mucus formation. (70)
Although it is not clear if nonspecific NSAIDs are associated with a loss of NO function, it is clear
that providing additional NO through nitrate and nitrite ameliorates the damage caused by NSAIDs.
Summary
Everyday lifestyle choices and the advents of modern pharmacology and standard of care negatively

6. affect NO production, activity, and homeostasis. As such, proposed pharmacological therapies
should consider NO levels as a safety and long-term efficacy indicator. Increasing awareness of
maintaining NO levels through diet, lifestyle, and caution regarding medication is critical for
physicians and patients. Medical literature is rich in establishing NO homeostasis as crucial for
cardiovascular, neural, and immune health, but this level of correlation is not reflected in modernday
medical practice. This article focused on raising awareness regarding the same. Perhaps the best
strategies for overcoming NO deficiencies from lifestyle or pharmacological therapies is through the
nitrate-nitrite-NO pathway. (18) This approach is dietary and might be the safest of ways to maintain
endothelial health and NO homeostasis in vivo. This simple dietary modification can prevent a
number of chronic disease states, including cerebral vasospasm, ischemia-reperfusion injury, sickle
cell disease, hypertension, peripheral artery disease, pulmonary hypertension, gastric ulceration,
inflammatory bowel disease, hyperlipidemia, and inflammation.n. (71), (72) A more holistic approach
to modern-day care, with an emphasis on maintaining NO levels, has future implications in reversing
the current trends seen in chronic disease management.
Notes
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[ILLUSTRATION OMITTED]
Conflicts of Interest
N. S. Bryan has a financial interest in NeoGenis Inc. as a paid consultant and stock ownership. N. S.
Bryan also has stock options in SAW Pharrna and has received honoraria for consulting services to

11. Bristol Myers Squibb. His financial and research conflicts of interest are managed by UTHSCH
Conflicts of Interest Management Plans, developed from and reviewed by the Re,.0.1rch Conflicts of
Interest Committee, and approved by the executive vice president for research at the University of
Texas Health Science Center at Houston.
Nathan S. Bryan, PhD
Assistant Professor of Molecular Medicine Texas Therapeutics Institute Brown Foundation Institute
of Molecular Medicine, Department of Integrative Biology and Pharmacology University of Texas
Graduate School of Biomedical Sciences at Houston University of Texas - Houston Health Science
Center
1825 Pressler St. 530C
Houston, Texas 77030
713-500-2439 office
713-500-2447 fax
Nathan.Bryan@uth.tmc.edu
http://www.uthouston.edu/imm
Nathan S. Bryan, PhD, is an assistant professor of molecular medicine within the Brown Foundation
Institute of Molecular Medicine, part of the School of Medicine at the University of Texas Health
Science Center at Houston. He is also on faculty within the Department of Integrative Biology and
Pharmacology and Graduate School of Biomedical Sciences at the UT Houston Medical School. Dr.
Bryan's research is dedicated to providing a better understanding of the interactions of nitric oxide
and related metabolites with their different biological targets at the molecular and cellular level and
the significance of these reactions for physiology and pathophysiology. Dr. Bryan has spent the past
12 years on research related to diagnosing NO insufficiency and natural strategies to restore NO
production in the human body. These fundamental discoveries may provide the basis for new
preventive or therapeutic strategies in diseases associated with NO insufficiency and new guidelines
for optimal health. Dr. Bryan has published a number of highly cited papers and authored or edited 4
books.
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