Functional Nitric Oxide Nutrition

Functional Nitric Oxide Nutrition:
Dietary Strategies to Prevent and Treat Chronic Disease

By Nathan S. Bryan, PhD
Copyright © 2018 by Nathan S. Bryan

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ii
Message from the Author
Click on the link below the image to hear a special message

from Dr. Bryan.
https://www.youtube.com/watch?v=LZisH_pjh7Y&t=3s
iii
Endorsement
Dr. Nathan Bryan and I have been educating doctors at the

same medical conferences for years, and have often spoken
together on various aspects of nitric oxide. He explains
beautifully the basics of what nitric oxide is, what it does
throughout the human body to maintain health, and how it is
manufactured. I have learned so much from Nathan, and his
lectures always spike my interest to further my understandings
and learn more about this amazing signaling gas, a gas that so
few doctors know much about. Nathan truly was the original
impetus to me to become an expert in nitric oxide.
As a women’s health specialist, I speak on the critically

important interplay between hormones, particularly estrogen
and nitric oxide. Estrogen is vital to having adequate levels of
nitric oxide in women. Without adequate nitric oxide, women
have problems with metabolic health, blood pressure control,
cognitive wellness, heart function, fertility, pregnancy, and
more. There is a beautiful synergy between estrogen and
nitric oxide. Without Nathan, I would not have become the
well-versed physician I am with this amazing topic. All of my
foundational knowledge of nitric oxide came from his teachings
and research.
Dr. Bryan is a true trailblazer in this still infant field of nitric

oxide, not only in researching all of the roles played by nitric
oxide but also in researching the treatment of its deficiency
states. He teaches the means to evaluate one’s level of nitric
oxide, the foundations of diet conducive to increasing nitric
oxide, proper oral hygiene to allow our food to generate nitric
oxide, the hazards of certain pharmaceuticals, the beneficial
impact of hormones, and the role of supplementation to
augment the body’s production of nitric oxide.
v
My friend and colleague, Dr. Bryan has made a huge impact
on the implementation of nitric oxide medicine to improve and
even save lives. This new book he has written is a gem. I read
and absorbed every word, and though I thought I would not
actually learn anything new, in fact I learned quite a bit of new
information from my read.
I am an integrative gynecologist and teach doctors around the

world on the newest concepts in integrative women’s health.
All I teach on nitric oxide has evolved from my learning from
Nathan. I owe him a huge debt.
There is no doubt that his new book on nitric oxide will become
a groundbreaking treatise on a little appreciated and vitally
important health topic. You’ve made a wonderful decision to
buy and read this book. Enjoy it as I did, and refer back to
it often. Nitric oxide should and will become an integral part
of your knowledge base and approach to the care of your
patients if you’re a doctor. Or, it will guide your personal health
choices if you’re looking to optimize your own health or that of
a loved one.
I celebrate the research Nathan has done in the field of nitric

oxide, and I congratulate you for what you are about to do –
join the world of nitric oxide literate people.
Felice Gersh, M.D.
vi
Table of Contents
Foreword................................................................................................. 3
Introduction............................................................................................. 5
1. History of Nutrition.......................................................................... 9
2. What is Nitric Oxide and How is it Produced?.......................... 17
3. How is Nitric Oxide Derived from Nutrients?............................. 27
4. Bacteria: Helping Us Do What We Cannot Do......................... 35
5. Focus on Nitrogen-Based Nutrients........................................... 45
6. Why Vegetables are Good for You............................................. 49
7. Undisputed Health Benefits......................................................... 55
8. But I Thought Nitrite and Nitrate Were Toxic............................ 63
9. Defining Nitrite and Nitrate as Nutrients.................................... 69
10. How Much Do You Need?........................................................... 79
11. Now What? Simple Steps to Regain your Health..................... 87
12. Looking Forward............................................................................ 97
About the Author................................................................................ 101
About Dr. Bryan’s Work.................................................................... 103
Other Books by Dr. Bryan................................................................ 105
Connect with the Author................................................................... 107
Acknowledgements........................................................................... 109
References......................................................................................... 111
Resources........................................................................................... 123
vii
Regarding health, wouldn’t it be better to build a fence at
the top of a cliff than park an ambulance at the bottom?
~ Dr. Dennis Burkett
1
Foreword
Nitric oxide (NO) is one of the most amazing and versatile cell
signaling molecules in mammalian biological systems, including
cardiovascular, immunology, neurology, gastroenterology,
endocrinology, pulmonary, musculoskeletal, and others. The
cardiovascular effects of NO are vasodilatation, reduction in
blood pressure, decrease in oxidative stress and inflammation,
reduction of vascular and cardiac smooth muscle growth,
inhibition of platelet aggregation, and leukocyte adhesion to the
endothelium, which decreases atherosclerosis and coronary
heart disease. Deficiencies of NO bioavailability are related to
hypertension, atherosclerosis, coronary heart disease, both
obstructive and vasospastic diabetes mellitus, dyslipidemia,
and stroke. In fact, NO addresses the three finite responses
to cardiovascular disease: inflammation, oxidative stress, and
vascular immune dysfunction.
Numerous blood tests and noninvasive cardiovascular

tests—such as asymmetric dimethyl arginine (ADMA) and
devices that measure endothelial function, respectively—can
access the bioavailability of NO, which is highly correlated
with endothelial dysfunction, the earliest predictor of future
cardiovascular events. NO also relates to arterial compliance
and elasticity, which predict future coronary heart disease
(CHD), stroke, and large and small arterial vascular wall
disease. This can be measured with computerized arterial
pulse wave analysis (CAPWA). Therefore, it is important
to provide adequate NO production via the eNOS/ arginine
pathway as well as the oral cavity of commensal organisms.
Normal stomach acid production and symbiosis that we have
with our oral microbiome provide the pathway for NO from
fruits and vegetables—especially dark green leafy vegetables
and beets—to convert nitrates to nitrites and then to NO. This
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NATHAN S. BRYAN
later pathway is most important in patients after the age of 40

years.
This new data has huge implications to developing treatment

strategies to lower heart disease and other modern diseases
associated with NO deficiency. The ability of the Mediterranean
and DASH diets (Dietary Approaches to Stop Hypertension) to
reduce CHD, diabetes mellitus, lower glucose, improve lipids,
and increase longevity are due to the high concentration of
nitrates in the food that are converted to nitrites and NO by
this system, the basis for functional nitric oxide nutrition.
Dr. Bryan has performed a masterpiece of literary genius in his

new book, “Functional Nitric Oxide Nutrition.” The discovery
of nitric oxide production in humans is considered one of the
most important medical discoveries of the last 100 years. This
book will take the reader on a simple and clear journey of
how to understand and use nitric oxide to maintain health and
longevity. The text takes us from basic science to clinical work
and therapeutics. Each chapter is well organized, and the flow
from one concept to the next is smooth and effortless for the
reader. Few books transform complexity to singular clarity
as this one does. I recommend this excellent book to all my
patients and to medical professionals who wish to enter into
an education of nitric oxide from one of the leading experts
and innovators in this area.
Mark C. Houston MD MS MSc FACP FAHA FASH FACN

FAARM ABAARM, ABCCH.
Associate Clinical Professor of Medicine
Vanderbilt University Medical School
Director, Hypertension Institute and Vascular Biology
Medical Director of Division of Human Nutrition
Saint Thomas Medical Group, Saint Thomas Hospital
Nashville, Tennessee
4
Introduction
Health care and medicine have been and continue to be

major issues in the U.S. The U.S. spends more money on
health care than any other nation, yet we rank last or close
to last for all major developed countries. These statistics are
staggering, considering we know a lot about the mechanisms
of disease. Scientific and medical discoveries in the past 100
years have led to enormous advancements in medical care
here in the U.S. and worldwide. Primarily through vaccines
and antibiotics/antiseptics, the burden of death from infectious
disease has drastically declined. The advent of medical devices
and advancements in emergency medicine has 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 many 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. All causes of death adjusted for age
decreased by 12.5 percent from 2000 to 2008. But the percent
of the population 18 years and over with heart disease has
risen from 10.9 percent to 11.8 percent, and the population
65 years and over has risen from 29.6 percent to 31.7 percent
over the same eight years. Diabetes has gone from 8.5 percent
of the population 20 years and older to 11.9 percent in just
eight years. The percentage of people with hypertension has
risen from 28.9 percent to 32.6 percent. Cancer has followed
a similar trend, increasing from 4.9 percent to 6.1 percent in
patients 18 years old and over.
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NATHAN S. BRYAN
These statistics suggest that although people are living longer,

they are not living better—or they are living with a chronic
disease that requires care and treatment. It is the care and
treatment of these chronic patients that causes the enormous
economic burden on the health care system and the patients.
In fact, from 2000 to 2008, total health care expenditures
increased from $1.1 to $2.0 trillion, or from $4,032 to $6,411
per capita, and today a decade later are over $10,000 per
capita. This highlights a very serious problem with our health
care system and clearly demonstrates that what we are doing
is not working. As Albert Einstein once said, “Insanity is doing
the same thing over and over and expecting different results.”
We can, and must, do better.
The top ten causes of death in the U.S. are heart disease,
cancer, chronic respiratory diseases, accidents/drugs
misuse and abuse, stroke, Alzheimer’s, diabetes, influenza/
pneumonia, kidney disease, and suicide. Eight out of these top
ten causes of death have a clear and indisputable mechanism
involving nitric oxide production. I am trained in biochemistry
and molecular and cellular physiology. I take great pride and
responsibility in the fact that I understand how the body works
and what goes wrong in people who get disease. What we
have learned in nitric oxide biochemistry and physiology over
the past twenty years allows us to very safely and effectively
restore nitric oxide production. At the time this book was
written, there are over 150,000 published scientific and medical
papers on nitric oxide revealing that if you can prevent loss of
nitric oxide production and availability, you can prevent many
age-related chronic diseases—including 80 percent of the top
ten causes of death.
The intentions of this book are to highlight new science that

is not considered, or even known, by most physicians and
healthcare practitioners, so patients and consumers can use
this information and new knowledge to take control of their
own health. My objectives are to educate readers on the
importance of nitric oxide, to clearly illustrate that diet and
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FUNCTIONAL NITRIC OXIDE NUTRITION
lifestyle modifications can significantly improve nitric oxide

production, and to arm the reader with information they can
begin to implement into their daily life. My hope is that people
will not only be able to improve their current health conditions,
but prevent many of these diseases in the first place. After
reading this book, I want everyone to walk away with these
basic learnings:
1. Nitric oxide is one of the most important molecules

produced in the human body. It controls and regulates
most cellular functions.
2. Loss of nitric oxide is the earliest event in the onset and

progression of most, if not all, chronic diseases.
3. As we age, we lose the ability to produce nitric oxide,

putting us at risk for age-related diseases.
4. New discoveries in science and medicine reveal what

goes wrong in people who can’t make nitric oxide. We now
know how to fix these basic problems with simple diet and
lifestyle changes, without drug intervention.
With these four basic concepts and by employing the strategies

I teach you in this book, you can make great progress to
improve your current health status, whether you are sick
and hoping to get better or currently healthy and just want to
prevent getting sick.
This book is organized to provide a historical account of

discoveries in nutrition so you can become aware that what
I am describing is not dissimilar to transformative health and
disease discoveries that occurred many years ago. I then
want to introduce the concept of how nitric oxide (NO) is
produced from nitrate and nitrite found in many of the foods
we eat. This will provide the background for why functional
nutrition focused on NO will transform public health. The next
few chapters provide some complicated steps and pathways
7
NATHAN S. BRYAN
that are necessary and sufficient for adequate NO production.

The role of bacteria is essential.
Next, I describe why vegetables are good for you. It goes

beyond simple antioxidants and the vitamins and minerals
they provide. Historically, we have been misinformed that
nitrite and nitrate are toxic food additives only found in hot
dogs and bacon. I will properly inform you on the true science
around nitrite and nitrate, and provide perspective on the
major sources of nitrite and nitrate in our diet. The following
chapters review the published scientific literature on how
much we need to get in our diet to achieve the clinical benefits
of consuming such amounts. The final chapters provide a
clear program and guidelines for what you can do to make
sure your NO levels remain adequate to support your own
physiology, whether you are a young, well-trained athlete or
a senior citizen wanting to improve your quality of life. Finally,
I will provide my perspective on where NO science may take
us in the future.
The field of nitric oxide is relatively new. The molecule was

only discovered in the late 1970s and early 1980s. In fact,
it is one of the newest discoveries in medicine. Very few
people, including physicians treating patients, know about
nitric oxide. My hope is that through this book and others,
nitric oxide will become as well-known as omega-3 fatty acids
or vitamin C. The science is clear that without sufficient nitric
oxide production, your body cannot and will not heal, nor will
it perform optimally. The single most important thing you can
do for your health is to improve or restore normal nitric oxide
production. This book will help you do just that.
8
-1-
History of Nutrition
Advancements in nutrition and discoveries of new nutrients

have had revolutionary effects on health and disease
management throughout history. The word nutrition itself
means the process of nourishing or being nourished, especially
the process by which a living organism assimilates food and
uses it for growth and replacement of tissues. A nutrient is
defined as any substance that nourishes an organism, and to
nourish is to sustain with food or nutriment—to supply with what
is necessary for life, health, and growth. Nutrients, therefore,
are substances that are essential to life that must be supplied
by food. Some categories of nutrients include water, protein,
carbohydrates, vitamins, minerals, fatty acids, and amino
acids. There are obviously many specific examples within
each of the categories. What is clear, though, is that nutrients
are fundamental to physiological systems, and good nutrition
can prevent many diseases. It follows that the absence of
nutrients can cause disease. We and others have recently
discovered a new set of nutrients, the lack of which may be
the cause of all chronic diseases—including cardiovascular
9
NATHAN S. BRYAN
disease, the number one killer of men and women worldwide.

To the contrary, sufficient ingestion or supplementation of
these specific nutrients may prevent many of these chronic
diseases.
Today more than ever, gaining nutrition knowledge can

make a transformative impact on our lives. Air, soil, and
water pollution, in addition to changing farming practices,
have depleted our soils of vital and essential nutrients and
minerals. The widespread use of food additives, chemicals,
sugar, and unhealthy fats in our diets contributes to many of
the degenerative diseases of our day such as cancer, heart
disease, arthritis, and osteoporosis. We know a poor diet
causes many health problems such as blindness, anemia,
scurvy, preterm birth, stillbirth, and cretinism. Diet and nutrient-
related issues can also lead to health-threatening conditions
like obesity and metabolic syndrome, and even common
chronic systemic diseases as cardiovascular disease,
diabetes, and osteoporosis. In order to appreciate the recent
discovery of a new nutrient that is essential for optimal health,
it is important to understand central historical milestones in
nutrition.
In 400 B.C., Hippocrates, the “Father of Medicine,” said to his

students, “Let thy food be thy medicine and thy medicine be thy
food.” Isn’t it ironic that we have abandoned food and nutrition
as a staple of modern medicine? In fact, although 25 hours
of nutrition instruction is recommended in the medical school
curriculum, many physicians report having enough nutrition
knowledge to counsel and treat their patients. Perhaps it is time
to get back to the basics, especially when modern medicine
fails us miserably today. This is apparent in the U.S., which
has been ranked as one of the lowest developed countries in
health care despite spending more than any other country.
In the same era as Hippocrates in 400 B.C., foods were often

used as cosmetics or medicines for the treatment of wounds.
In some of the early Far Eastern biblical writings, there were
10
references to food and health. One story describes the

treatment of eye disease, now known to be due to a vitamin
A deficiency, by squeezing the juice of liver onto the eye.
Vitamin A is now known to be stored in large amounts in the
liver, so this is the basis for the successful treatment.
During the 1500s, scientist and artist Leonardo da Vinci

compared the process of metabolism in the body to the burning
of a candle. This revealed that he had at least a conceptual
understanding of how our body utilizes nutrients to fuel energy
production. In 1747, Dr. James Lind, a physician in the British
Navy, performed the first scientific experiment in nutrition. At
that time, sailors were sent on long voyages for years and
they developed scurvy (a painful, deadly, bleeding disorder).
During this time, only nonperishable food products such as
dried meat and breads were taken on the voyages, since
there was no refrigeration and fresh foods would spoil. In his
experiment to try and figure out how to treat these sailors,
Lind gave some of the sailors sea water, others vinegar, and
the rest limes. Only those given the limes were saved from
scurvy. We now know that the vitamin C in the limes is what
prevented the scurvy. However, vitamin C wasn’t discovered
until the 1930s, or almost 200 years after Lind’s experiment.
11
NATHAN S. BRYAN
Lind didn’t know it at the time, but he had discovered a vital

nutrient.
In 1790, George Fordyce recognized calcium as necessary for

fowl survival. In the early 1800s, it was discovered that foods
are composed primarily of four elements—carbon, nitrogen,
hydrogen, and oxygen—and methods were developed for
determining the amounts of these elements. Justus Liebig of
Germany, a pioneer in early plant growth studies, was the first
to point out the chemical makeup of carbohydrates, fats, and
proteins. Carbohydrates were made of sugars, fats were fatty
acids, and proteins were made up of amino acids. Just before
the turn of the 20th century, in 1897, Christiaan Eijkman, a
Dutchman working with natives in Java, observed that some of
12
the natives developed a disease called beriberi, which caused

heart problems and paralysis. He recognized that chickens,
when fed the native diet of white rice, also developed the
symptoms of beriberi. When he fed the chickens unprocessed
brown rice (with the outer bran intact), they did not develop
the disease. Based on his observations in chickens, Eijkman
then fed brown rice to his patients and they were cured of
beriberi. This was another important case demonstrating that
specific nutrients found in certain foods could cure disease.
Nutritionists later learned that the outer rice bran contains
vitamin B1, also known as thiamine, a deficiency of which is
the cause of beriberi.
In the early 1900s, E.V. McCollum, while working for

the U.S. Department of Agriculture at the University of
Wisconsin, developed an approach that opened the way to
the widespread discovery of nutrients. He discovered the first
fat soluble vitamin, vitamin A. He found that rats fed butter
were healthier than those fed lard, as butter contains more
vitamin A. About the same time, Dr. Casmir Funk was the
first to coin the term “vitamins” as vital factors in the diet. He
wrote about these unidentified substances present in food,
which could prevent the diseases of scurvy, beriberi, and
pellagra (a disease caused by a deficiency of niacin, vitamin
B-3). The term vitamin is derived from the words vital and
amine, because vitamins are required for life and they were
originally thought to be amines—nitrogen compounds derived
from ammonia. We will discuss nitrogen based nutrients in
subsequent chapters.
In 1925, E.B. Hart discovered that trace amounts of copper are

necessary for iron absorption. In 1927, Adolf Otto Reinhold
Windaus synthesized vitamin D, for which he won the Nobel
Prize in Chemistry in 1928. In 1928, Albert Szent-Györgyi
isolated ascorbic acid, and in 1932 proved that vitamin C
prevented scurvy. In 1935, he synthesized it, and in 1937, he
won a Nobel Prize for his efforts. In the 1930s, William Rose
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NATHAN S. BRYAN
discovered the essential amino acids, the building blocks of

protein.
In 1968, Linus Pauling, a Nobel Prize winner in chemistry,

created the term orthomolecular nutrition. Orthomolecular is,
literally, “pertaining to the right molecule.” Pauling proposed
that by giving the body the right molecules in the right
concentration (optimum nutrition), people could use nutrients
to achieve better health and prolong life. There is substantial
scientific and clinical evidence to support that the right
nutrients are found in the right concentrations in whole foods
(when grown in nutrient dense soils). Therefore, consuming
a balance of the right foods containing different essential
nutrients and vitamins should be able to prevent and/or treat/
cure many diseases. Pauling himself said that most diseases
are caused by nutrient deficiencies.
My research program was the first to demonstrate the absence

of nitrite and nitrate in the diet made many disease conditions
worsen, including injury from heart attack. We were also the
first to demonstrate that supplementing or restoring these
missing nutrients back into the diet, reversed the disease and
injury and could actually prevent chronic inflammation (main
driver of disease). These were some of the first discoveries in
this era of “functional nitric oxide nutrition”.
These discoveries over the years have led many scientists

and pharmaceutical companies to begin to isolate active
compounds from foods and synthesize them to sell as vitamins
or dietary supplements, or create derivatives they could then
patent. It is clear from history that these active compounds
can cure, treat, and even prevent disease. The timeline below
chronologically illustrates how these discoveries have led
to amelioration of many diseases and the beginning of the
functional nitric oxide nutrition era. The following chapters will
highlight how these handful of discoveries have the potential to
change the face of medicine and health care. Simply restoring
functional nutrients that are missing in much of the population
14
and certainly missing in sick people can restore cellular

function and improve human health. History has a pattern
of repeating itself and has demonstrated unequivocally that
giving back a missing nutrient has profound effects on human
health and disease.
If that’s the case, if nutrients really do have impact on human

health and disease, you might be wondering why vitamins,
nutrients, and supplement packaging labels include a phrase
saying that their products and information are not intended
to diagnose, treat, cure, or prevent any disease. These also
usually state that their health claims have not been evaluated
by the Food and Drug Administration (FDA). In October of
1994, the U.S. Congress passed the Dietary and Supplement
Health and Education Act (DSHEA). It sets forth what can
and cannot be said about nutritional supplements without
prior FDA review. Many believe this is advocated by large
pharmaceutical companies to protect their “drugs” for the
treatment of disease. If drug companies have competition
from vitamins or supplements, then this will obviously affect
their profits. In January of 2000, the FDA clarified that
supplement makers can state their products can improve
the structure or function of the body or improve common,
minor symptoms. Examples of allowable statements include
“maintains a healthy heart,” “helps you relax,” supports healthy
digestion,” “is good for symptoms of PMS,” “strengthens joint
structure,” etc. Overall, due to this law, vitamin, herb, and
nutrient manufacturers have greater freedom to say what their
products can do to improve our health.
While this law limits what vitamin manufacturers can claim

about preventing or curing diseases, its passage has been a
major milestone in the natural health field. It acknowledges
the millions of people who believe dietary supplements can
improve their diets and bestow good health. It opens the way
for people to obtain the information they need to make the
best nutritional choices. It is also very important for there to
be some safety and oversight on nutritional supplements and
15
NATHAN S. BRYAN
vitamins so consumers can be certain they are actually taking

and getting what the product label indicates.
Understanding how nutrients and active molecules have been

discovered from foods even thousands of years ago—before
we had any of the advanced technology and analytical tools
we have today—provides a strong foundation for the next
chapters. Moving forward, we’ll uncover a very important,
newly discovered nutrient that may be able to cure, prevent,
and treat the number one killer of men and women worldwide:
cardiovascular disease. This new nutrient may very well be the
most important nutrient ever discovered. Imagine a nutrient
that could prevent heart disease, Alzheimer’s, diabetes, and
chronic infections. Keep reading, because the next chapters
will not only describe this nutrient, but also provide the robust
scientific evidence that it can indeed prevent, treat, or cure all
of the above.
16
-2-
What is Nitric Oxide and
How is it Produced?
Imagine, for a moment, a single molecule that can dramatically

improve your health—and you can maintain normal levels of it
within your body simply through diet and lifestyle. This single
molecule can: prevent high blood pressure (hypertension);
combat a disease that damages your heart, brain, and
kidneys; keep your arteries young and flexible; prevent, slow,
or reverse the buildup of artery-clogging arterial plaques; help
stop the formation of artery-clogging blood clots (the result
of plaques bursting and spilling their contents into the blood
stream); and lower triglycerides. By doing all of the above,
this molecule can also reduce your risk of heart attack and
stroke—the first and fifth killers of men and women worldwide.
That in itself would be pretty remarkable, but this molecule can

additionally reduce the risk of diabetes and disastrous diabetic
complications such as chronic kidney disease, blindness,
hard-to-heal foot and leg ulcers, and amputations. It also has
17
NATHAN S. BRYAN
the ability to limit the swelling and pain of arthritis, boost the
power of pain-relieving drugs, reverse erectile dysfunction
(ED), calm the choking inflammation of asthma, protect your
bones from osteoporosis, help provide the mood-lifting power
behind antidepressant medications, assist the immune system
in killing bacteria, and limit skin damage from the sun.
A lack or deficiency of this molecule is what causes most

diseases, including cardiovascular disease, the number one
killer of men and women worldwide. That is why the scientific
and medical community is so excited that this molecule can be
restored and optimized through nutrition from specific foods
and diet. As 1998 Nobel Laureate Louis J. Ignarro, Ph.D.,
said: “There may be no disease process where this miracle
molecule does not have a protective role.”
You can stop imagining this miracle molecule now, because

it’s real and accessible. What is it? Nitric oxide—otherwise
known (by its chemical formula) as NO.
What is nitric oxide? It is a signaling molecule. In fact, it is how

cells in our body communicate with one another. Nitric oxide
is NO, one atom of nitrogen and one atom of oxygen—simple
as can be. So simple, in fact, that it’s a gas when it is produced
within the body. When it’s created and released by cells, this
gas easily and quickly penetrates nearby membranes and
other cells, sending its signals. In less than a second, NO
signals:
• arteries to relax and expand

• immune cells to kill bacteria and cancer cells
• brain cells to communicate with each other
In fact, NO sends crucial signals within every cell, tissue,

organ, and system of the body. This all occurs when our body
produces nitric oxide upon demand. We now know that there
are two pathways to make NO in the human body.
18
The endothelial cells
These are cells that line all our blood vessels throughout the
circulatory system, and they generate NO from L-arginine. In
young, healthy blood vessels, this pathway is functional and
generates sufficient NO to maintain normal blood pressure
and the integrity of the circulatory system.
Dietary nitrate and nitrite consumption
Nitrate is found primarily in green leafy vegetables and root

vegetables. It is broken down into nitrite and nitric oxide based
on bacteria that live in and on our body.
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NATHAN S. BRYAN
The endothelial pathway (pathway 1) becomes disrupted with

age. There are conditions where either one or both pathways
become disrupted. If one system goes down, the other one
can compensate and pull the weight for the other. However,
if both systems fail, then you are in trouble and disease will
start to set in. The older you are, the lower your NO, because
of the age-related decline in endothelial function. We lose
about 10 to 12 percent of our endothelial-produced NO per
decade. In fact, by the time we are about 40 years old, we
have lost about 50 percent of our ability to generate NO from
our endothelial cells.
In one study, Italian researchers evaluated forearm blood

flow—the standard measurement of endothelial health—in 47
people with normal blood pressure and 49 people with high
blood pressure. They found that in both groups, those who
were older had poorer endothelial-dependent vasodilation—
the NO-sparked ability of arteries to widen and permit health-
giving blood flow. That weakening of the endothelium was in
perfect parallel to aging; decade by decade, NO-powered,
endothelial-dependent vasodilation declined. Specifically:
• 30 years old and younger

Endothelial-dependent vasodilation was strongest.
• 31 to 45 years old
Vasodilation was 11 percent weaker than in the 30-and-
younger set.
• 46 to 60 years old
Vasodilation was 13 percent weaker than in the 31 to
45-year-olds.
• 60 and older
Vasodilation was 28 percent weaker than in the 46- to
60-year-olds.
20
All in all, those 60 and older had vasodilation that was 52 percent
weaker—less than half as strong—as those 30 and younger.
And these were older people who did not have high blood
pressure. Having high blood pressure actually accelerates
this entire process. Therefore, effectively managing your
blood pressure is the single most important thing you can do
to protect your blood vessels and the function of all organs,
tissues, and cells.
“Advancing age is an independent factor leading to

the progressive impairment of endothelium-dependent
vasodilation in humans,” conclude researchers in the Science
journal Circulation, which is published by the American Heart
Association. The functional changes in the endothelium
precede the structural changes seen in vascular disease by
many years, sometimes decades. But why do blood vessels
become dysfunctional with age?
According to the researchers, dysfunction occurs because

of “A progressive reduction of NO availability.” In fact, their
findings suggest that “in aged individuals NO availability is
21
NATHAN S. BRYAN
almost totally compromised.” In a similar study, Japanese

researchers tested vasodilation in 18 healthy people, aged
23 to 70. The patients’ responses to the vasodilator is
striking, showing a near-perfect correlation between age and
endothelial health. A 23-year-old in the study had an artery
that expanded more than five times its width when given a
vasodilator. The artery of the 70-year-old expanded a little
more than two times. The researchers concluded: “Coronary
blood flow response to acetylcholine (an endothelium-
dependent vasodilator) decreased significantly with aging.”
Why? Because of the age-related decrease in the release

of “endothelium-derived relaxing factor or NO.” Specifically,
another study by the same team of Japanese researchers
found a loss of 75 percent of endothelium-produced NO in
people 70 to 80 years old as compared to 20-year-olds.
It’s important to emphasize that this decline happens not only

to people with CVD, but in healthy older adults too. These are
people who don’t have high blood pressure, high cholesterol,
or circulation-damaging diabetes. In other words, it happens
to everybody who gets older. It follows that if NO is so critical
for optimal health and disease prevention, if we could figure
out how to prevent the decline in NO production with aging,
then perhaps we could prevent many age-related diseases.
This would truly be the “holy grail” in cardiovascular medicine.
With more than 150,000 papers published on NO, the scientific

and medical literature tells us that loss of NO production and
availability is the earliest event in the onset and progression of
cardiovascular disease. In fact, this molecule is so important
that the scientists who discovered it were awarded a Nobel
Prize in Medicine or Physiology in 1998. Years prior in 1992,
NO was proclaimed “Molecule of the Year” by Science
Magazine. Dr. Valentin Fuster, the former President of the
American Heart Association and the head of cardiology at
Mount Sinai Hospital in Manhattan, says that “the discovery
22
of nitric oxide and its function is one of the most important in

the history of cardiovascular medicine.”
NO does more than just regulate our cardiovascular system.

Our immune system is very intimately related to NO production.
During the past two decades, NO has been recognized as
one of the most versatile players in the immune system. It is
involved in the pathogenesis and control of infectious diseases,
tumors, autoimmune processes and chronic degenerative
diseases. To regulate immune responses, NO will kill off
invading pathogens from bacteria, to viruses on the one hand
and on the other hand modulate immunosuppression during
tissue-restoration and wound-healing processes. Much of
these effects comes from the effects NO has upon immune
cells.
NO is also important in stem cells. Mesenchymal stem

cells are immune modulators. They will help suppress the
inflammatory response that the body produces in many
different conditions. Nitric oxide works hand in hand with
mesenchymal stem cells and macrophages to make the stem
cell environment more conducive for stem cell repair. There
is essentially no pathological condition in the body where NO
does not play a role in the management of the condition. This
is another example of how losing the ability to make NO will
hinder your body’s ability to heal and fight off infections.
Since NO production becomes compromised with aging,

older people must then rely on consuming nitrate in their
diet to achieve an optimal level of NO in their system. Under
ideal conditions, both pathways—endothelial production and
dietary NO consumptions—provide about 50 percent of our
total body NO. Thus, when both are working and functioning
properly, we make sufficient NO and all the systems in our
body are functioning properly. Scientific research has figured
out how to “fix” the problem of lost NO from pathway 1 with
age, and it has discovered how to optimize pathway 2 so that
23
NATHAN S. BRYAN
no matter how old people get, they can still have sufficient NO
production to become resistant to disease.
Since NO is a gas that is gone in less than a second after it

is produced within the body, it is not simple to supplement
the body with NO gas. Therefore, an understanding of how
the body makes NO is required, so we can then provide the
body with the raw material and nutrients it needs to efficiently
produce NO. Nutrients, remember, are substances that are
essential to life and must be supplied by food. Since NO itself
cannot be delivered in foods, we must identify the precursors
(i.e. the building blocks) of this molecule that become the
nutrients necessary for NO production: nitrite and nitrate.
What we now know about NO may explain a very early concept

around what “animates” humans. The Roman physician
Galen based his description of the vascular system on the
concept of “pneuma,” or spirits—a vital principle consisting of
matter in a finely divided or ethereal state that flowed through
the vascular and nervous systems and animated the entire
organism. Many diseases were thought to owe their origin to
some disturbances of these ethereal spirits. This paradigm
actually prevented the advancement of medical science for
centuries. But today, evidence on the production and effects
of such an ethereal substance, NO, does exist. Nitric oxide
is indeed formed in many organs and has important roles in
physiology and pathophysiology.
Even earlier in Ancient Chinese medicine and culture, the

concept of Qi (pronounced chi) was introduced. “Qi” (氣)
literally means gas in Chinese. Chinese medicine has long
recognized that gasses in the body have important functions
such as: warming, energizing, and communicating with
metabolic functions (See table below). Medically, there are
names for many kinds of gasses based on their actions in
the body. While they lacked the technology to identify and
measure the exact gasses, Chinese physicians were keenly
aware of how these gasses permeated the body and the
24
points at which they exited the skin. Nitric oxide has been
suggested to be Qi. It can pass freely through membranes,
and transmit signals from neurons to target cells. When NO
is out of balance, bad things typically happen. Nitric oxide
has well-defined functions. It sends messages to tell cells
how much energy they should be producing. When the right
proportion of NO reaches fat stores, they begin to transform
fat into useable energy and heat.
25
NATHAN S. BRYAN
Fundamental medical and physiological properties of

molecules described centuries ago are consistent with what
we know today about NO. We now have a firm understanding
and convergence of many ancient and effective medical
strategies that revolve around NO. We now know that we
can improve the production of this molecule from foods and
nutrients that many people are missing. It appears that we
may have identified a critical component of our diet that many
people are missing. In fact, certain things we have been
taught to fear and avoid in our diet may be saving our lives
from inflammatory diseases.
26
-3-
How is Nitric Oxide Derived
from Nutrients?
Have you ever wondered why salads are served before the
main course? Natural social behaviors, although we don’t
always think about it, exist for biochemical and physiological
reasons. If they didn’t, they would work themselves out of
societal behavior. Since the late 1970s, we knew that dietary
nitrate found primarily in green leafy vegetables (salads)
would be absorbed in our gut and then recirculated and
concentrated in our salivary glands. At that time, it was not
known why our bodies would do this—especially since at that
time nitrate was considered a precursor to nitrosamines that
could cause cancer. Well, advance the science forty years and
we now have a clear understanding of why nitrate from green
leafy vegetables is absorbed and concentrated in our saliva.
It is nature’s way of protecting us from post-prandial (after a
meal) inflammation or the damage and oxidation caused from
digesting and breaking down complex foods such as proteins,
fats, and carbohydrates that happens after we digest salad
27
NATHAN S. BRYAN
prior to the meal. This occurs through a human nitrogen cycle,

called the enterosalivary circulation of nitrate.
Since nitric oxide is so critical to our health and our bodies

can produce it from specific nutrients in the foods we eat,
nature designed an exquisite system utilizing the bacteria in
our mouth (the oral microbiome) to provide a source of NO
from the foods we eat. Research has shown that critically ill
patients who are intubated and fed through a feeding tube
do not get any nitric oxide produced from this pathway since
these processes prevent saliva flow, chewing, and swallowing
saliva. Furthermore, people who use mouthwash disrupt this
pathway and become deficient in NO. More on this later.
Nitrate found in salad greens or most leafy green vegetables

can be metabolized by certain and select bacteria that live on
the back part of our tongue. When we chew and eat slowly,
this allows the nutrients in the food to reach the crypts of the
tongue for the bacteria to reduce (break down the nitrate
molecule) to nitrite and nitric oxide. Since this first part of
digestion is only occurring for a period of 10 to 20 seconds at
a time, there is limited opportunity for all the nitrate to reach
the bacteria. Then, when we swallow the chewed up food, it is
in the stomach for several minutes (45 to 120, depending on
a number of factors) where it is broken down further. Food is
then emptied into the upper part of the small intestines called
the duodenum. It is here where nitrate is selectively taken
up across the lining of the gut and then recirculated to the
salivary glands. Each time we salivate, the nitrate in our saliva
is broken down to nitrite and NO.
28
This is illustrated below:
This process occurs slowly over the course of several hours,

so that each time we salivate for the next few hours, the nitrate
is utilized by bacteria that live in the crypts of the tongue as an
energy source and in the process the bacteria generate nitrite.
Nitrite and nitrate are distinctly different molecules, although
they sound nearly the same and most people confuse them for
one another. Nitrate and nitrite in the saliva are as much as
100 times higher than in the circulation. Each time we swallow
our saliva, it reaches the stomach—which is very acidic—and
the nitrite in saliva becomes nitric oxide gas. However, this is
dependent upon sufficient stomach acid production. People
who take proton pump inhibitors (Prevacid, Prilosec, etc.) for
29
NATHAN S. BRYAN
reflux disease disrupt this pathway. We will talk about this in

more detail later.
The nitric oxide produced from this pathway has many

beneficial functions besides dilating blood vessels internally.
It is this nitric oxide gas that is generated from salivary nitrite
that helps kill foodborne pathogens such as E.coli, Listeria,
Chlostridium botulinum. Not only does it kill off bad bacteria
that may cause sickness, but it also kills off Helicobacter
pylori, the bacteria that is responsible for gastric ulcers. NO
generated from swallowing our own saliva is also responsible
for increasing blood flow to the stomach to help with the
absorption of nutrients, and to promote the integrity of the
protective lining of the stomach to prevent damage from acid.
We now know how each step in this process can become
disrupted and what the consequences are in specific patients.
The production of nitric oxide from swallowing our own saliva

is dependent upon stomach acid production. The production
of stomach acid may be the most important process for the
health of all humans. The nitrite that is concentrated in our
saliva from the action of nitrate-reducing bacteria becomes
nitric oxide gas when the pH falls below 4. As long as the
inside of the stomach is acidic, NO is generated each time
we swallow. Patients taking proton pump inhibitors (PPIs or
acid reflux medications) intentionally inhibit stomach acid
production; therefore, the pH of the stomach is no longer
acidic and no nitric oxide is produced. It is now clear that
patients who have been taking PPIs for 3 to 5 years have a
30 percent higher incidence of heart attack and stroke. This
is due to insufficient nitric oxide production. Can you begin to
recognize the importance of NO? If its synthesis is shut down,
you increase your risk of heart attack and stroke. Independent
of nitric oxide production, stomach acid is required for the
breakdown of proteins we consume in our diet. The enzymes
in our stomach that break down protein into amino acids
are only active when the stomach is acidic. When there is
insufficient stomach acid production from PPI use, proteins
30
are not broken down completely and are then undigested

protein fragments (peptides) that are absorbed across our gut.
These peptides are then recognized by our immune systems

as a foreign substance and this activates our immune system.
This is the reason for food-borne allergies and many auto-
immune diseases. With proton pump inhibitors being the
third largest class of prescribed drugs worldwide, millions of
people take them—generating tens of billions of dollars in
revenue for pharmaceutical companies. The inhibition of nitric
oxide production combined with auto-immune conditions and
insufficient nutrient absorption make these drugs potentially
the most dangerous drugs on the market for long-term use.
However, many people become dependent upon these drugs
and are unable to stop taking them. Obviously, this would be
best—but for those who cannot, utilizing functional nitric oxide
nutrition can partially overcome the inhibition caused by these
drugs. Therefore, for those patients taking antacids, they must
incorporate some form of nitric oxide nutrition that can provide
a source of NO under these conditions. The last chapter will
illustrate how to do just that.
In order to understand what goes wrong in people who can’t

make NO, we must break down each step in the pathway.
The first step in the entero-salivary circulation of nitrate and
production of nitric oxide is to make sure we get enough nitrate
from our diet through consumption of green leafy vegetables.
The next step involves the activation of nitrate into nitrite and
nitric oxide by bacteria. Nitrate is inert in the human body,
meaning that humans cannot utilize this molecule since we
do not have the enzyme systems necessary to activate it.
The process of converting nitrate to nitrite and nitric oxide is
dependent upon select and specific bacteria that live in and
on our body—at least a dozen or more types, according to
research.
It has also been shown that people who do not have these
bacteria in their mouth become nitric oxide deficient, and are
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NATHAN S. BRYAN
thus at increased risk for developing cardiovascular disease.

In fact, it has been shown that if you use a mouthwash that
kills oral bacteria for seven days, your blood pressure will go
up. This is profound. Who would have thought that mouthwash
could affect your blood pressure? The observation that killing
oral bacteria causes an increase in blood pressure reveals
without a doubt that there are specific bacteria that are helpful
and beneficial in generating nitric oxide. So, if you are taking
an antiseptic mouthwash, my recommendation is that you
stop. It is better to have bad breath than to have a heart attack.
Statistics based on the U.S. Census data and Simmons
National Consumer Survey (NHCS) show that 188.2 million
Americans used mouthwash/dental rinse in 2011. This figure
was projected to increase to 206.35 million in 2020.
This suggests that more than half of the U.S. population may
not be getting a physiological response from dietary nitrate
consumption, either from consuming green leafy vegetables
or from drinking nitrate-enriched beetroot juice. To put this
clearly, one can never get all the health benefits of eating a
good diet rich in green leafy vegetables if these bacteria are
lacking. From the previous chapter, we know that many of
the health-promoting benefits of green leafy vegetables are
due to their nitrate content. However, if we do not have the
right bacteria to activate the nitrate into a usable form for nitric
oxide generation, then we will never get all the health benefits
of vegetables. We will still get the other nutrients and vitamins
that vegetables provide—but without the nitric oxide benefit,
we lose many of their heart-healthy properties.
The many health benefits of this newly discovered pathway

are clear. Eating vegetables or consuming beetroot juice
has been shown to lower blood pressure, improve exercise
performance, improve blood vessel function and elasticity,
improve cognition in aging patients with pre-Alzheimer’s
disease or vascular dementia, and basically combat all aspects
of aging. Interestingly, all these effects are lost if the subjects
do not swallow their own saliva or if nitrate is removed from
32
the vegetables or beet juice. Remember when your mother

told you as a child to eat your vegetables and do not spit?
This was actually very good advice, since this will promote
NO production. This is clear evidence of the importance of
nitrate in our diet and having the right systems functional in
our bodies to be able to utilize nitrate to generate nitric oxide.
To summarize, vegetables that contain dietary nitrate that

may be converted to nitric oxide in the body. However, this
requires a certain threshold of nitrate and the right oral nitrate-
reducing bacteria to reduce nitrate to nitrite. Once nitrite is
concentrated in the saliva and swallowed, it generates nitric
oxide in the acid environment of the stomach. Therefore,
vegetables can be a source of nitric oxide, but require all three
phases to be efficient. Below are limitations and problems with
just using vegetables to restore nitric oxide:
1. All vegetables aren’t created equal. Depending on what

type of vegetable and where the vegetables are grown and
the soil conditions, they may or may not contain sufficient
nitrate for nitric oxide production. Nitrate content of many
vegetables has been measured and quantified, and some
contain no nitrate at all. Since there is no standardization
of nitrate in vegetables, just consuming any vegetable
does not guarantee you will be getting nitrate.
2. Lack of oral nitrate-reducing bacteria. Based on our

studies, we estimate that 30 to 40 percent of the population
does not have the right oral nitrate-reducing bacteria.
This is due primarily to use of antibiotics and antiseptic
mouthwash. Also, people with poor oral hygiene do not
appear to have the right bacteria—likely due to being out-
competed for resources by disease-causing bacteria.
3. Lack of stomach acid production. Most people take

an antacid medication that suppresses stomach acid
production. There is also an age-related decline in stomach
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NATHAN S. BRYAN
acid production due to insufficient zinc and iodine in the

diet or uptake in the stomach.
While there are limitations, we have shown that simply

providing more nitrate through the diet is sufficient for
changing the oral microbial communities and providing a
more favorable environment for them to grow. Having the
right nitrate-reducing bacteria will generate nitrite and nitric
oxide. Most, if not all, pathogenic bacteria are sensitive to NO.
Therefore, restoring the oral cavity with good nitrate-reducing
bacteria will help eradicate the bad bacteria.
You should also make sure the vegetables you eat contain
sufficient nitrate. Vegetables like kale, spinach, arugula,
beets, cabbage, and many other green leafy vegetables
typically have the highest nitrate content. Making sure you
have sufficient stomach acid is another critical consideration.
Taking a teaspoon of apple cider vinegar prior to meals will
help acidify the stomach and lead to better digestion and
more NO production from your diet. Apple cider vinegar is
acetic acid that has a pH of around 2. You need a stomach pH
of less than 3 in order to optimize NO production. If you are
taking antacids, make every effort to stop and get off these
medications. They were never designed to be used daily or
for more than a couple of days at a time. As you will learn,
anything that disrupts nitric oxide production is very damaging
to your health. To summarize:
1. Eat more green leafy vegetables.
2. Stop using mouthwash and don’t overuse antibiotics.
3. Make sure your stomach is making stomach acid.
These three simple lifestyle modifications will significantly

improve your nitric oxide production.
34
-4-
Bacteria: Helping Us Do
What We Cannot Do
Most people think of bacteria as infectious disease causing

organisms. While there are some harmful bacteria that can
make humans sick, most of the bacteria that live in and on
our body are essential for our own health. In fact the number
of bacteria that live in and on our body outnumber human
cells by a factor of ten. Yes, there are over 10 times more
bacteria cells than our own cells. These bacteria are what are
referred to as symbiotic bacteria, meaning that they benefit
and the human host benefits from their metabolic activity.
In essence, they do essential biochemistry or perform
reactions that humans cannot do thereby providing essential
nutrients or molecules for normal human physiology. The
human microbiome is composed of many different bacterial
species, which outnumber our human cells ten to one and
provide functions that are essential for our survival. Most of
the research over the past couple decades has focused on
the gut microbiome. The bacteria of the lower intestinal tract
35
NATHAN S. BRYAN
play an essential role in maintaining a healthy body. In fact,

many people take a probiotic to help restore gut bacteria,
to aid in digestion and even combat a number of diseases.
These bacteria are necessary for nutrient acquisition and bile
acid recycling, among other activities. Less studied are the
oral bacteria. It has been known for many years and even
decades that oral pathogens related to periodontal disease
and gingivitis can also cause cardiovascular disease. As
discussed in previous chapters, a human nitrogen cycle has
been identified. This pathway, termed entero-salivary nitrate-
nitrite-nitric oxide pathway, can positively affect nitric oxide
production and represents a potential symbiotic relationship
between oral bacteria and their human hosts—meaning that
the bacteria are performing essential metabolic steps that we
as humans cannot perform.
As a result, both the bacteria and the human hosts benefit.

The oral commensal bacteria provide an important metabolic
function in human physiology by contributing a source of nitric
oxide. As we learned earlier, NO is one of the most important
molecules produced in the human body. This bacterial process
is analogous to the environmental nitrogen cycle whereby soil
bacteria convert atmospheric nitrogen from fertilizers to usable
forms for plant growth. Human nitrate reduction requires the
presence of nitrate-reducing bacteria, as mammalian cells
cannot effectively reduce this anion. The discovery of the
NO pathway in the 1980s represented a critical advance in
understanding cardiovascular disease, and today a number of
human diseases are characterized by NO insufficiency. There
is sufficient and convincing evidence in the literature that these
bacterial communities provide the host a source of NO that
may be able to overcome insufficient NO production from the
blood vessels. The focus of this chapter is to discuss the new
science of oral bacterial nitrate reduction, providing humans
with a rescue pathway for conditions of NO insufficiency and
diseases associated with NO insufficiency.
36
Nitrate that is concentrated in our saliva after we eat a meal

with green leafy vegetables or beetroot juice has to be
metabolized to nitrite, a reaction that human cells are unable
to perform. Manipulation of the bacteria as a therapeutic target
for disease management is on the near horizon. In fact, you
have probably heard of fecal transplants, where doctors can
take bacteria from the fecal matter of a healthy person and
transplant it into the colon of a sick person with great success
and change the bacteria profile from “bad” to “good”. I predict
this will be similar for oral bacteria, whereby people with good
oral microbiome can donate a portion of their bacteria to be
transplanted or inoculated into those people who may not
have the right oral bacteria. The mouth cavity is an attractive
target for probiotic and/or prebiotic therapy because of the
ease of access.
Although a few nitrate-reducing bacteria in the oral cavity

have been identified, we are just now beginning to identify
which bacteria are necessary and sufficient for nitric oxide
production. Everyone has a different microbiome, and
specific bacteria are associated with good nitrate reduction.
These bacteria are: Granulicatella adiacens, Haemophilus
parainfluenzae, Actinomyces odontolyticus, Actinomyces
viscosus, Actinomyces oris, Neisseria flavescens, Neisseria
mucosa, Neisseria sicca, Neisseria subflava, Prevotella
melaninogenica, Prevotella salivae, Veillonella dispar,
Veillonella parvula, and Veillonella atypica. Additionally,
Fusobacterium nucleatum and Brevibacillus brevis were
designated as species of interest even though they are
typically at much lower abundance.
So, how do you know if you have the right bacteria? This is
a fundamentally important question to ensuring your own
health and preventing many age-related diseases due to nitric
oxide deficiency. Since nitrite accumulates in saliva from the
reduction of nitrate in the oral cavity, determining salivary
nitrite concentrations may offer simple means to determine
the presence or absence of nitrate-reducing bacteria.
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NATHAN S. BRYAN
However, understanding that nitrite in our saliva comes from

NO produced within the lining of our blood vessels and also
from reduction of nitrate by oral bacteria. There are steps in
this pathway that can become disrupted and lead to changes
in salivary nitrite. Each step is described below:
1. Nitrate (from oxidation of NO or diet) uptake in the gut and

transport to salivary glands. This is dependent upon how
much nitrate you get from your diet and how much NO
your body makes.
2. Nitrate secretion by salivary glands: The volumes of saliva

produced vary depending on the type and intensity of
stimulation.
3. Oral bacterial nitrate reduction: Humans lack a functional

nitrate reductase, so salivary nitrate reduction is dependent
upon oral commensal nitrate-reducing bacteria.
4. Oral pH: Healthy oral pH is between 6.5 and 7.5. The pKa
of nitrite is 3.4, which means that any pH around or lower
than 3.4 nitrite will become NO. Therefore, any condition
that lowers the pH in the oral cavity may destabilize nitrite
and affect the use of salivary nitrite as a measure of nitrate
reduction. We need an acidic environment in the stomach
but not in the mouth.
There are now salivary test strips that measure the amount
of nitrite in your saliva. Salivary nitrite is a biomarker for total
body nitric oxide bioavailability. If your saliva is low in nitrite,
then your body is low in nitric oxide. It could be that your blood
vessels aren’t making enough NO, or it could be that you
don’t have the right bacteria to metabolize nitrate to nitrite.
Regardless of the issue, if you are low in salivary nitrite, then
your body is low in NO. So, grab a test strip and apply your
saliva to the test pad. Within a couple of seconds, the test strip
will turn a shade of pink. The darker the pink/red color, the
more NO you have. The lighter shade of pink is representative
38
of low NO. If you test low, then go eat a salad—preferably

a high nitrate salad with spinach, kale, etc. Approximately
90 to 120 minutes later, retest using the salivary test strip.
If you improve the color on the test strip, then you have the
right bacteria to convert the nitrate in the salad into nitrite and
nitric oxide in your body. This is a good thing. If you do not
improve the color on the test strip, then you do not have the
right bacteria to convert the nitrate in your diet into nitric oxide.
If the latter is the case, what do you need to do? First, if you
are taking a mouthwash, you should stop. Second, you should
consume more nitrate-
containing foods. The
nitrate-reducing bacteria
require nitrate in order to
respire and colonize in
the oral cavity. If you do
not eat enough nitrate-
rich vegetables, then you
are not providing enough
fuel for these bacteria to
survive.
As a result of this human

nitrogen cycle and enterosalivary circulation of nitrate (both
from diet and endogenous NO production), and subsequent
reduction to nitrite in the mouth, sampling salivary nitrite can
be used as an accurate representation of the presence or
absence of nitrate-reducing bacteria. Saliva offers a number
of advantages as a biological compartment for diagnostics.
There is sufficient evidence to show that increased circulating
levels of plasma nitrite correlate with changes in blood
pressure. Furthermore, blood and salivary levels of nitrite
increase after a nitrate load, and killing the oral bacteria with
a mouthwash or antibiotic causes a decrease in salivary nitrite
and an increase in blood pressure. To the contrary, as NO
availability is decreased, both plasma and salivary levels of
nitrite decline. So, whether there is sufficient NO produced
39
NATHAN S. BRYAN
by the blood vessels (pathway 1, or the L-arginine pathway)

to form nitrite and nitrate in the circulation—which is then
concentrated in our salivary glands—or there is sufficient
nitrate ingested in the diet (pathway 2, nitrate-nitrite-nitric
oxide pathway), this will be reflected as nitrite in the saliva.
Understanding the basis and rationale for sampling salivary
nitrite as well as recognizing the limitations, physicians and
patients can gain new information from this non-invasive
diagnostic, including accurate assessment of total body NO
availability, cardiovascular risk, and NO homeostasis. More
research is needed in order to determine if this approach may
have clinical utility. Salivary nitrite measurements may offer
an indirect measure of the ability of humans to reduce nitrate.
The realization that bacteria in our mouth can affect our

systemic health has been known for a while, but the realization
that it is nitric oxide that may be responsible for this effect is
groundbreaking. The potential to exploit the symbiotic nitrate-
nitrite-NO pathway to NO production is profound, particularly
because adequate and sustained control of blood pressure
is achieved in only about 50 percent of treated hypertensive
patients—including all classes of anti-hypertensives
medication. Since NO is known to affect many other biological
processes, this knowledge also has the potential to affect all
chronic diseases. As cardiovascular disease remains the
top killer in the U.S., accounting for more deaths each year
than cancer, designing new diagnostics, treatments, and
preventives for diseased and at-risk individuals is essential.
This poses the question - Do people that have high blood
pressure, simply have an oral microbiome problem? With
over 50% of the people taking anti-hypertensive medications
getting no response from these medications, this new concept
may explain why. It’s time to focus on the problem.
Additionally, because NO is an important signaling molecule in

all body systems, exploiting the oral microbiome to contribute to
NO production and maintain NO homeostasis has the potential
to affect human health beyond the cardiovascular system.
40
Being able to repopulate essential nitrate-reducing bacteria

in the oral cavity is of immense interest, since this may be an
effective means to restore NO availability in the human body.
Recent research in rats found that simply feeding nitrate in
the diet could lead to a significant increase in nitrate-reducing
Haemophilus parainfluenzae. Additionally, Granulicatella and
Aggregatibacter, which have both been associated with poor
oral health in humans, decreased with additional nitrate in the
diet. These results suggest that high nitrate diets may induce
changes in oral microbiome communities to more efficiently
reduce nitrate to nitrite and NO, which could be beneficial
both for reducing blood pressure and inhibiting bacterial
species associated with poor oral health. Since nitrite and NO
are toxic to many pathogenic bacteria, simply adding more
nitrate in the form of green leafy vegetables may allow for
restoring the balance of good vs bad bacteria. This suggests
that dietary nitrate and nitrite may act as a prebiotic for the
oral microbiome.
For the past thirty years, scientists have focused on NO

production/regulation at the level of nitric oxide synthase
(NOS), pathway 1 described earlier from L-arginine. However,
this pathway becomes dysfunctional with age and disease and
produces less and less NO the older and sicker we get. The
notion that this can be overcome by targeting oral bacteria is
profound and revolutionary. Therapeutically, then, perhaps an
effective strategy to promote NO production and overcome
conditions of NO insufficiency may not be targeted at pathway
1 in the blood vessels, but rather on targeting specific oral
nitrate-reducing bacterial communities. Understanding and
harnessing this redundant compensatory pathway may prove
to be a viable and cost-effective strategy. Furthermore, new
research reveals that this may in fact be the biochemical and
physiological link between oral health and cardiovascular
disease through maintenance of NO production. Because
NO signaling affects all organ systems and almost all disease
processes described to date, this novel approach to NO
41
NATHAN S. BRYAN
regulation has the potential to affect the study and treatment

of many diseases across all organ systems.
What is clear from this most recent research is that disruption

of nitrite and NO production in the oral cavity may contribute to
the oral-systemic link between oral hygiene and cardiovascular
risk and disease. The identification of new biomarkers for
NO insufficiency and the exploitation of the oral microbiota
to increase cardiovascular health will be enabled by further
characterization of the enzymatic activities of native oral
bacterial communities from larger groups of people and specific
patient populations. These groups should consist not only of a
specific U.S. population, but also of others (European, Asian).
It is likely that the oral microbiomes of different ethnic groups,
even those within different regions of the U.S., vary widely. It will
be important to determine whether different nitrate-reducing
communities are more prevalent in geographically dispersed
healthy populations; likewise, it will also be important to
determine whether different nitrate-reducing communities are
lacking in specific patient populations from around the world.
If certain patient populations lack specific nitrate-reducing

bacteria, personalized treatments to enrich for nitrate reducers
may be warranted. Is it tempting to wonder whether the use of
mouthwash may be discouraged as part of such treatments.
Indeed, studies have shown that using mouthwash raises
blood pressure in humans. Additionally, while antibiotics
are sometimes used to target specific bacterial species, it is
possible that potential deleterious effects of antibiotic usage
on nitrate-reducing communities may preclude the use of
antibiotics in specific patient populations.
Clearly, the potential for the entero-salivary nitrate-nitrite-

NO pathway (pathway 2) to serve as a NO bioavailability
maintenance system by harnessing the nitrate reductase
activity of specific commensal bacteria calls may be profound
and truly transformative. Future studies are likely to unveil new
paradigms on the regulation and production of endogenous NO
42
that are likely to be new targets for specialized, multi-faceted,

and potentially personalized therapeutic interventions. These
studies will have the potential to: 1) redefine the meaning of
“healthy oral microbiome” to include microbes associated with
NO production, 2) provide a new target for NO-based therapies
and open a new direction in cardiovascular research, and 3)
allow development of new diagnostics targeted at specific
oral microbial communities or select bacteria, the absence
of which may reflect a state of NO insufficiency and change
the treatment strategies for NO restoration in a number of
different diseases. This is the focus of my current research
program. With the loss of NO signaling and homeostasis being
one of the earliest events in the onset and progression of
cardiovascular disease, targeting microbial communities early
in the process may lead to better preventative interventions
in cardiovascular medicine. This may also affect the way oral
health professionals recommend hygienic practices.
43
-5-
Focus on Nitrogen-
Based Nutrients
The atmosphere and environment on Earth is made up of 78

percent nitrogen (N2). Nitrogen is a vital macronutrient for
plants, necessary for basic cellular function and production
of many basic cellular components, such as DNA, RNA,
and proteins. It is also an essential nutrient for plant growth,
development, and reproduction. Plants need nitrogen in a form
that is usable by the plant for growth, since they are unable to
utilize elemental nitrogen or N2. Nitrogen itself is not directly
available to plants, but some can be converted to available
forms by microorganisms found in the soil or from decaying
matter. Despite nitrogen being one of the most abundant
elements on Earth, nitrogen deficiency is probably the most
common nutritional problem affecting plants worldwide, and
perhaps nitrogen in the form of nitrate or nitrite may be the
most common nutritional problem affecting humans worldwide.
This is because both plants and humans must have the right
form of nitrogen.
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NATHAN S. BRYAN
Soil nitrogen exists in three general forms: organic nitrogen

compounds, ammonium (NH4+) ions, and nitrate (NO3-) ions.
At any given time, 95 to 99 percent of the potentially available
nitrogen in the soil is in organic forms, meaning decaying
plant and animal residues. Nitrogen in the form of nitrate or
ammonia is taken up by plants through roots from inorganic or
organic sources, such as amino acids. In agricultural settings,
nitrogen may be a limiting factor for plant growth and yield.
That is why nitrogen-based fertilizers are added to crops and
fields. A plant deficient in this nitrogen will shunt resources
away from its shoot in order to expand its root system and
acquire more nitrogen. Most plants take nitrogen from the
soil continuously throughout their lives, and nitrogen demand
usually increases as plant size increases.
A plant supplied with adequate nitrogen and has the

capabilities to fix and assimilate nitrogen into ammonia. Nitrate
causes plants to grow rapidly and produces large amounts of
succulent, green foliage and vegetables. Adequate nitrogen
allows an annual crop, such as corn, to grow to full maturity
on time. A nitrogen-deficient plant is generally small and
develops slowly because it lacks the nitrogen necessary to
manufacture adequate structural and genetic materials. It is
usually pale green or yellowish because it lacks adequate
chlorophyll. Older leaves often become necrotic and die as
the plant moves nitrogen from less important older tissues to
more important younger ones. In nature, wildfires, although
devastating at times, do provide nature’s way of putting
nitrogen back into the soil. Farmers often burn crops and hay
fields to naturally replenish nitrogen making for beautiful lush,
green grass & crops. Again, nature has a tendency to take
care of itself.
46
Early experiments in herbivores in the 1830s proposed that

the relative nutritional values of plant foods could be assessed
from their contents of nitrogen. The scientists concluded
that foods that do not contain some form of nitrogen cannot
continue to support life. Most plants have bacteria or fungi
in the root systems that can fix nitrogen into usable forms
for energy. For plants that do not have a symbiont partner
to provide them with fixed nitrogen, nitrate, found in soil, is
the preferred source of fixed nitrogen. Therefore, any plant
or vegetable grown in soil accumulates primarily nitrate, but
also to a lesser extent nitrite. So, when we eat vegetables, we
consume nitrate, an essential nutrient. Although plants can
use nitrate as a form of energy, humans cannot make use
of nitrate. It must first be metabolized by bacteria that live in
and on the human body. This was discussed in the previous
chapter.
Nitrogen fixation is a reversible process that can be harnessed

based on specific needs of plants and also humans.
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NATHAN S. BRYAN
As you can see from above, all these forms of nitrogen can be
formed and utilized by plants and humans, once we consume
vegetables that have one or more forms of nitrogen. Different
vegetables store and accumulate more nitrate than others.
For example, kale, spinach, and beets typically contain high
amounts of nitrate, whereas lettuce, asparagus, and broccoli
contain less—although all green leafy vegetables contain
some level of nitrate and in some cases nitrite.
Humans require nitrate and nitrite from plant sources in order

to make nitric oxide. Just as in plants, humans require sources
of nitrogen to make amino acids. These form proteins, to make
DNA and to maintain normal metabolism. Therefore, nitrogen-
based nutrients in the form of nitrate and nitrite are essential
for human health and disease prevention.
48
-6-
Why Vegetables are
Good for You
We have been told since we were kids to “Eat your vegetables.”

Our moms and grandmas insisted we eat vegetables, and
most kids hate vegetables. However, your mom was right. We
should eat our vegetables, and we should eat them at every
meal. So, we have established that vegetables are good for
you. But perhaps we have missed the most important reason
vegetables are good for you. It is because they provide the
human body with nitrate and nitrite, which we discussed in
the previous chapter. There are scientific truths and facts that
cannot be denied or ignored. Any diet that is not based on sound
scientific evidence or rationale will always come and go and
be a “fad”. Diets and foods are the most important determinant
of your health. Nothing affects your health more than what you
eat. Our body is designed to get all the nutrients and material
it needs from our diet. Diets that eliminate certain food groups
that provide essential nutrients will never be sustaining. To the
contrary, diets that provide a balance of nutrients to replete
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NATHAN S. BRYAN
what the human body is missing will be the most nutritious

and helpful for human health. There are a number of diets that
have proven to be healthy, and their common denominator
is that they provide sufficient nitrite and nitrate for optimal
wellness. These include the Mediterranean diet, South Beach
Diet, Dietary Approaches to Stop Hypertension (DASH), and
Paleo. All of these diets recommend consumption of several
servings of vegetables, particularly green leafy vegetables, in
their meal plans. So what is it about these diets that allows
them to survive the test of time and remain at the top of the list
of the best diets out there?
The DASH diet is a clinically researched diet rich in fruits,

vegetables (especially green, leafy ones), low-fat dairy foods,
and with reduced saturated and total fat that can substantially
lower blood pressure. This diet is commonly used by those
interested in supporting healthy blood pressure levels through
the food they eat. The Mediterranean diet recommends
emulating how people in the Mediterranean region have
traditionally eaten, with a focus on foods like olive oil, fish
and vegetables. U.S. News and World Report called the diet
a “well-balanced eating plan” and pointed to research that
suggests the diet helps prevent some major chronic diseases
and increases longevity. The heart-healthy nature of all these
diets are due to the fact that they provide more dietary nitrite
and nitrate than the standard Western diet. We have analyzed
the foods from all these diet plans and found that one can
easily meet and exceed the accepted daily intake of nitrite
and nitrate.
Epidemiology is the branch of medicine that deals with the

incidence, distribution, and possible control of diseases and
other factors relating to health. Nutritional epidemiology is
an area of epidemiology that involves research to examine
the role of nutrition in the etiology of disease, monitor the
nutritional status of populations, and develop and evaluate
interventions to achieve and maintain healthy eating patterns
among populations. Most, if not all, epidemiological studies
50
investigating green leafy vegetables show that these foods

protect from cancer, cardiovascular disease, Alzheimer’s
disease, and many other chronic diseases. For years,
scientists believed this association was due to the rich source
of antioxidants and vitamins vegetables provide. However,
when clinical studies are conducted investigating the individual
vitamins and antioxidants found in these vegetables, they
fail to reproduce the protective effects of the vegetables
themselves. Therefore, there must be an “unknown nutrient” in
these vegetables that is responsible for the protective effects.
A meta-analysis is a statistical technique for combining

the findings from independent studies. Meta-analysis of
antioxidant supplements affecting a number of diseases reveal
there is no benefit in fighting most diseases. Meta-analysis
is most often used to assess the clinical effectiveness of
healthcare interventions. It does this by combining data from
two or more randomized control trials. The current evidence
does not support the use of antioxidant supplements in the
general population or in patients with various diseases.
So, what is it about vegetables that clearly show protection
from cardiovascular disease, cancer, Alzheimer’s, and most
chronic diseases?
Is nitrate the answer?
That’s the question an international team of scientists from

the world-famous Karolinska Institute in Sweden and from
Boston University School of Medicine asked themselves
while wondering why vegetables protect the heart from
disease. Their scientific paper in the medical journal Nitric
Oxide—“Cardioprotective effects of vegetables: Is nitrate the
answer?”—offers this perspective:
1. Eating a diet rich in vegetables lowers blood pressure

almost as much as treatment with a standard pressure-
lowering drug.
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NATHAN S. BRYAN
2. The high content of nitrate in certain vegetables, and

its conversion to nitrite and NO, is the real reason why
vegetables are cardioprotective.
3. In fact, this pathway of NO generation works better than

the L-arginine pathway, which “malfunctions” in people
with heart disease.
4. Vegetarians (who have low rates of heart disease) consume

about 10 times more nitrate than nonvegetarians, as do
people who eat a Mediterranean-style diet, also shown to
protect against heart disease.
5. The task for scientists is to find the optimal level of nitrate

and nitrite intake for cardioprotection.
Their conclusions:
1. “The protective effect of certain vegetables on the

cardiovascular system is related to their high content of
nitrate.”
2. “The mechanism involves reduction of dietary nitrate to

nitrite [and] nitric oxide.”
3. “A continuous intake of nitrate-containing food such as

green leafy vegetables may ensure that tissue levels of
NO . . . are maintained.”
4. “If proven true, these considerations could have a profound

impact on our view of the role of diet . . . in the . . . prevention
of cardiovascular disease.”
They took the words right out of our mouths!
The evidence now supports the fact that nitrate and nitrite
may be the active nutrients in vegetables that make them so
healthy. Unlike vitamins and antioxidants like vitamins C, E,
52
and A, which have failed to show positive effects in clinical

trials, studies over the past fifteen years investigating nitrite
and/or nitrate have undeniably demonstrated that these
nutrients can in fact protect from heart disease, lower blood
pressure, improve athletic performance, improve Alzheimer’s,
and more. Several studies reveal that a single serving of leafy
green vegetables each day may help keep dementia away.
In one study, researchers evaluated the eating habits and
mental ability of more than 950 older adults for an average
of five years. They found that those who consumed one or
two servings of foods such as spinach, kale, mustard greens,
and/or collards daily experienced slower mental deterioration
than those who ate no leafy greens at all. The researchers
suggested that it may be due to the vitamin K provided
specifically by these leafy greens. However, I do not think it
is a coincidence that these are the foods most enriched in
nitrate.
Diets rich in fruits and vegetables are consistently associated

with a decreased risk of cancer. The U.S. federal government
has embraced this tenet as evidenced in the Healthy People
2000 and Healthy People 2010 campaigns, which advocated
the consumption of five or more servings of fruits and vegetables
daily. In addition, the National Cancer Institute (NCI), in
conjunction with the Produce for Better Health Foundation,
implemented the National 5 A Day for Better Health Program
in 1991 to encourage Americans to eat five or more servings
of fruits and vegetables every day in the context of a healthy
diet. These initiatives have led to Americans consuming more
fruits and vegetables, but they are not regularly consuming
the particular fruits and vegetables that are likely to impart
robust health effects. Specifically, they are not eating enough
dark green leafy vegetables, those with high amounts of
nitrate and nitrite.
What is clear from nutritional epidemiology is that people who

eat lots of green leafy vegetables are typically healthier than
those who do not eat green leafy vegetables. What then is
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NATHAN S. BRYAN
the active nutrient or compound that could account for the

impressive health benefits of vegetables? The answer is
clearly and undeniably nitrate. The previous chapters have
revealed how nitrate is taken up and then metabolized into
nitrite and nitric oxide by oral nitrate-reducing bacteria and
stomach acid production. So, to this point, we know that nitric
oxide is absolutely essential for optimal health and disease
prevention. We know nitrogen in the form of nitrate in our
foods is good, if we have the right oral bacteria to metabolize
nitrate into nitrite and nitric oxide. So, what do the published
clinical studies tell us on what we can expect from functional
nitric oxide nutrition? The next chapter will reveal just that.
54
-7-
Undisputed Health Benefits
Sufficient nitric oxide production is crucial for the maintenance

of every organ system in the human body, and the body
cannot function optimally without it. In fact, every single
chronic disease involves loss of nitric oxide production.
Whether it is glaucoma, macular degeneration, vascular
dementia, Alzheimer’s disease, heart disease, kidney disease,
liver disease, diabetes, stroke, or even cancer, all of these
conditions are characterized by a loss of sufficient blood flow
to the respective organs or tissues that does not allow them
to function properly. As a result, they fail. This then creates a
very simple model for treating, curing, and preventing most if
not all chronic diseases. Restore blood supply to the organs by
fixing the nitric oxide production pathways, so blood vessels
then have a way to dilate and bring new oxygen and nutrients
to these starved tissues. The million-dollar question, then, is
how do we do this?
When our body makes nitric oxide, nitrite is the main stable
product of NO in plasma. The amount of nitrite in the blood
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NATHAN S. BRYAN
reflects acute and chronic changes in NO production. Although

it is very difficult to measure NO directly in the blood, increases
in nitrite in the blood after an increase in NO production is an
effective way to measure one’s ability to produce NO from
the L-arginine pathway. Therefore, people who are deficient
in NO are deficient in nitrite. It has been shown that exercise
increases nitrite in healthy people. To the contrary, older
unhealthy individuals—who are unable to produce NO when
they exercise—fail to increase nitrite and also fail an exercise
stress test. This is diagnostic for heart disease. Under fasting
conditions, the ability to increase plasma nitrite in response
to exercise actually predicts how well one can perform . Age-
dependent alterations of the structure and function of blood
vessels predispose older individuals to increased risk of
cardiovascular disease.
Similarly, if your body can’t make NO when you begin to

exercise, this is a critical determinant of your risk for heart
disease. This usually becomes evident in people ages 60 and
over when they go for a routine exercise stress test. Similarly,
when you engage in sexual activity, you need an increase in
blood flow to the sex organs to get and maintain an erection.
This is true for both men and women. So, if your body cannot
make NO before or during sexual activity (which is physical
exercise), then you develop sexual dysfunction or erectile
dysfunction. Again, this is diagnostic for more serious vascular
concerns that indicate your body does not make sufficient NO.
This can occur much earlier, sometimes in late 30s and early
40s. With over 50 percent of men over the age of 40 showing
symptoms of some degree of erectile dysfunction, this is the
first sign of insufficient NO production and allows time for
nutritional intervention. Functional nitric oxide nutrition can be
a very effective strategy to overcome these age-related and
other disruptions in vascular NO production.
This raises the question as to whether providing more nitrite

and nitrate in the diet can restore NO-based signaling and
functions. The answer to this question has transformed
56
science and medicine over the past twenty years. These

discoveries have shed new light on how the human body
makes NO, and more importantly provided safe and effective
dietary strategies (functional nitric oxide nutrition) to restore
NO insufficiency. Dietary nitrite and/or nitrate can provide a
reservoir of NO activity and the ability to produce NO from the
blood vessels.
Hopefully by now you can appreciate the importance of NO

and the serious health consequences that occur when your
body cannot make sufficient NO. The concept of functional
nitric oxide nutrition can provide the human body with a
source of NO that it otherwise cannot produce, and also fix
the underlying problems of why people cannot make sufficient
NO. Drug therapy does not do this. Only functional nutrition
can do this. Simply providing more nitrite and nitrate in the diet,
and fixing the broken systems that allow for the metabolism
of nitrate to nitrite and nitrite to NO, has shown remarkable
results in human clinical trials. Over the past ten years,
there have been many published studies showing health
benefits in humans supplementing dietary nitrite and nitrate,
including blood pressure regulation and sports performance.
For the majority of studies in humans, investigators used
beetroot juice—standardized to a known amount of nitrate.
The findings show that increasing plasma nitrite levels can
increase NO production and improve oxygen efficiency and
athletic performance.
In studies in older adults, nitrate supplementation with beetroot

juice reduced blood pressure and positively influenced exercise
capacity. Even in patients with peripheral artery disease,
nitrate supplementation improved exercise performance,
whereas L-arginine was ineffective in these patients. This
demonstrates that the nitrate-nitrite-nitric oxide pathway can
rescue and overcome disruptions in the L-arginine pathway.
Dietary nitrate can also lower blood pressure. Drinking a bottle
of beetroot juice containing nitrate lowers blood pressure six
hours later in healthy adults.
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NATHAN S. BRYAN
Antiseptic mouthwash eradicates the benefits of nitrate
When relying on nitrate alone from dietary sources, there are

a number of steps in the process that can become disrupted
and will not allow the nitric oxide mediated benefits from nitrate.
All the health benefits of nitrate are completely abolished if
people taking nitrate are not allowed to swallow or if they use
an antiseptic mouthwash to kill oral bacteria. Studies have
shown that use of antiseptic mouthwash for seven days caused
a decrease in salivary and plasma nitrite, with an increase
in systolic and diastolic blood pressure, demonstrating the
removal of these bacteria with an antibacterial mouthwash
will very likely block the NO-dependent biological effects of
dietary nitrate.
Over 180 million Americans use mouthwash on a daily

basis, and in 2015 alone, approximately 269 million antibiotic
prescriptions were dispensed from outpatient pharmacies in
the United States—enough for five out of every six people
to receive one antibiotic prescription each year. Interestingly,
at least 30 percent of these antibiotic prescriptions were
unnecessary. Use of both antiseptic mouthwash and antibiotics
disrupts the oral microbiome and leads to a complete lack of
nitrate reduction, or at least a decreased efficiency of nitrate
reduction and conversion to nitrite.
Also, given the diversity and variability of the oral microbiome

between certain individuals and cultures, it is uncertain how
many people have the correct nitrate-reducing bacteria. With
prevalence of antibiotic and antiseptic mouthwash use in the
U.S. along with periodontal disease and poor oral hygiene, it
would not be surprising if over half of the population is unable
to reduce dietary nitrate. This means that although they may
be consuming what is considered a healthy diet even with
sufficient nitrate, they are unable to get a nitric oxide benefit
due to lack of nitrate reduction and conversion to nitrite by
bacteria. This should be a new consideration in patient
assessment.
58
Stomach acid is required for optimal NO production
Even if you have the right oral nitrate-reducing bacteria, this

does not always mean you will get a benefit from the nitrate
consumed in your diet. Stomach acid is required for optimal
effects of salivary nitrite. Nitrite concentration in the saliva
from reduction of dietary nitrate when swallowed becomes
protonated (nitrite pKa ~ 3.4) to form nitrous acid and nitric
oxide. Proton pump inhibitors (PPIs), a type of antacid used
by people with acid reflux, shuts down NO production by
inhibiting stomach acid production and increasing gastric
pH—which will prevent formation of nitrous acid from inorganic
nitrite, and, in turn, NO release. Indeed, studies have shown
that taking antacids will block the blood pressure lowering
effects of orally administered sodium nitrite. Furthermore,
PPIs blunt the favorable effects of antioxidants on nitrite-to-
NO conversion in the stomach. PPIs also specifically lead to
the accumulation of asymmetric dimethyl L-arginine (ADMA).
ADMA is generated during metabolism of cellular proteins

consumed through the diet. ADMA is broken down by the
enzyme dimethylarginine dimethylaminohydrolase, found
in many cells. The inhibition of the DDAH enzyme is the
major contributor to increases in ADMA in animal models
and patients with cardiovascular risk factors. Evidence now
demonstrates that PPI drugs directly inhibit DDAH activity.
In addition to inhibiting DDAH, PPIs affect the amount of the
enzyme that normally produces NO that is present in cells.
So, they actually decrease the amount of NO the body can
produce. Altogether, these findings provide direct proof that
antacid drugs decrease nitric oxide production.
Studies published several years ago reveal that people who

have taken antacid drugs for three to five years had about 30
percent more heart attacks and strokes. This is a huge effect.
Imagine all the people who could benefit from this information.
There are approximately 64.6 million prescriptions written for
gastroesophageal reflux disease (GERD) medications in the
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NATHAN S. BRYAN
United States on an annual basis, accounting for over $11

billion in total healthcare expenditures in the U.S.—and this
does not even include the over the counter (OTC) market.
Any therapy that increases stomach pH will interrupt NO
generation from salivary nitrite. Clear evidence is emerging
that PPIs have adverse cardiovascular effects. These
effects may be mediated primarily or at least in part through
a disruption in NO production/signaling. They should be
considered when PPIs are prescribed, especially in patients
at increased cardiovascular risk.
It is important at this point to explain the difference in nitrate

and nitrite. Nitrate is inert in humans, meaning that humans
cannot metabolize this molecule and if it is not first metabolized
by bacteria, then the body will just excrete nitrate through
the sweat, urine, and feces. Although humans rely solely on
bacteria to reduce nitrate to nitrite, we do have systems in
place to generate nitric oxide from nitrite. Studies conducted
by my research group in collaboration with Dr. David Lefer
were the first to show that supplementing nitrite and/or nitrate
in the diet protects the heart from injury from a heart attack.
Not only can it protect the heart from injury, but research has
shown it can protect the liver, the kidney, the brain, and most
organs. We were also the first to show that nitrite in the diet
can suppress inflammation, one of the hallmarks of all chronic
diseases.
Nitrite prevents the oxidation of lipids, which is critically

important in the development of heart disease. Nitrite in saliva
that is then swallowed is important in controlling foodborne
infection and in maintaining a healthy stomach lining. Long-
term dietary nitrite causes formation of new blood vessels,
which is important for people with peripheral artery disease
and conditions of low blood flow such as diabetes. Nitrite in
the form of an orally disintegrating tablet—with natural product
chemistry to reduce nitrite to NO—has been shown to modify
most if not all cardiovascular risk factors in people over the
age of 40, reduce blood pressure, and reduce markers of
60
inflammation. Since a substantial portion of steady state nitrite

concentrations in blood and tissue are derived from dietary
sources, supplementing nitrite in the diet can provide a first
line of defense for conditions associated with NO insufficiency.
61
-8-
But I Thought Nitrite and
Nitrate Were Toxic
Sometimes when we hear information repeated often enough,

we start to believe it—whether we intend to or not. Remember
being cautioned not to sit too close to the television or it would
ruin your sight? How about the warning not to swallow gum
because it would stay in your body for three years? While
intuitively we know that we need to let these notions go,
there’s something about these repeated warnings that causes
doubt to linger.
As a physiologist, I interact with a lot of people about how we

can maintain healthy bodies. Increasingly, I hear misguided
claims about food—like the idea that nitrite needs to be
avoided or that the best way to avoid it is to stop eating
cured meats like hot dogs and bacon. In some respects, it’s
understandable. People have heard cautions about nitrite for
decades, especially claims that nitrite could cause cancer.
But what people don’t understand is its critical role not only
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NATHAN S. BRYAN
in preventing foodborne illnesses, but most recently its

recognized health benefits due to its ability to form nitric oxide
or NO. Up until the 1970s, it was thought that nitrite and nitrate
were synthetic unnatural molecules, but we soon realized that
nitrite is actually produced in the bodies of mammals through
normal metabolic processes. This realization is what ultimately
led to the discovery of nitric oxide. Three American scientists
were awarded the 1998 Nobel Prize for the discovery of nitric
oxide. Now we know and appreciate the essential nature of
both nitric oxide and nitrite in human health and disease.
The claim that nitrite and nitrate are toxic and should be avoided
is particularly frustrating to me, because I know through my
work and the work of others that much of what is reported
in the previous chapters is true—that not only are nitrite and
nitrate safe, but absolutely essential for life. Published studies
reveal that these can protect from injury from heart attack and
stroke, prevent inflammation from a poor diet, and even lower
blood pressure, the primary risk factor for the development of
cardiovascular disease. In fact, there are currently twenty-six
clinical trials completed or ongoing using nitrite as a therapy
for conditions like heart failure, organ transplantation, cystic
fibrosis, and even leg ulcers just to name a few. Obviously, the
scientific and medical community understand the importance
of nitrite for human health—but the media sometimes mislead
us or sensationalize certain stories.
Still, claims that “no nitrite is added” appear increasingly on

foods these days, as if to suggest there is something to fear.
There’s just one catch: cured meats must contain a form of
nitrite to be cured, or ham and salami would just be, well . .
. pork. Most often, nitrite takes the form of celery powder or
celery juice. Sometimes its sea salt or even beet juice, which
are all rich sources of nitrate and nitrite. That’s right—nitrite is
found naturally in sea salt and most vegetables.
Now, here’s the real shocker for most people: Less than five
percent of the nitrite we consume comes from cured meats.
64
Approximately half of our daily nitrite intake comes from fruits,

vegetables, and human saliva. The other half of our body’s
nitrite exposure comes from the production of nitric oxide, one
of the most important molecules our body makes. Nitric oxide
is then metabolized to nitrite. So, our body makes nitrite as
part of its normal, healthy nitrogen cycle.
Studies have now shown that the health benefits of a

Vegetarian diet, the Mediterranean diet, the Japanese diet,
and the DASH (Dietary Approaches to Stop Hypertension)
diet are due to the nitrite and nitrate found naturally in the
foods consumed through these dietary patterns. Nitrite is also
found naturally in breast milk, nature’s most perfect food.
Everything we know about nutrition and dietary patterns, from
nursing infants to adults, demonstrates that nitrite and nitrate
are absolutely essential for health and wellness. People who
get more nitrite and nitrate from their diet have less disease.
People who do not get sufficient nitrite and nitrate from their
diet, or are unable to make nitric oxide, are more susceptible
to disease.
Where did the nitrite controversy originate? A single study

in the late 1970s claimed to have found increased tumors in
lab rodents who were given nitrite along with a chemical that
could react with nitrite (that chemical, by the way, is never
found naturally in our food supply). The study’s findings
were never confirmed in follow-up research. However, given
the controversy, the U.S. government’s National Toxicology
Program completed a study in 2000 in which rats and mice
were fed nitrate and nitrite in their drinking water. Recipe for
cancer? Not at all. The results published in 2001 concluded that
there was no evidence of carcinogenicity (or cancer-causing
activity) by nitrite at any dose. In fact, there was actually a
decrease in the incidences of mononuclear cell leukemia in
male and female rats. The researchers also concluded there
was no issue at all with nitrate or nitrite in causing cancer.
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NATHAN S. BRYAN
A panel that reviewed the NTP study’s findings voted and

concluded that based on the data, nitrite is not a carcinogen at
the levels used. The levels used in the study were far greater
than what could ever be consumed in the diet, but still showed
no evidence of cancer. That’s why nitrite is NOT on the list
of carcinogens maintained by the U.S. Certainly, there have
been studies that report nitrite-containing foods like bacon
and salami pose a health risk, but these are the same type
of studies that ask people to recall what they ate and then
analyze the data. They don’t occur in laboratories where
intake can be monitored and controlled. Remember, also,
that people consume diets—not single foods—and memories
about what was eaten are notoriously inaccurate.
The emergence and prevalence of “dietary nitrate” in products

within the marketplace has created a lot of confusion. The
confusion arises because for the past fifty years, we have been
told to avoid nitrate-containing products such as cured and
processed meats. So now, we are supposed to supplement
our diet with nitrate? This transformation is called scientific
progress. We know more now than we did fifty years ago. The
truth is that over 80 percent of our dietary nitrate comes from
green leafy and root vegetables. Only about 5 percent comes
from cured and processed meats, and the other 15 percent
comes from swallowing our own saliva. In fact, the reason
vegetables are good for us is partly due to their dietary nitrate
content and its metabolism to nitric oxide.
However, because there are biologically plausible mechanisms

whereby nitrate or nitrite can cause nitrosamines, there are very
effective strategies and mechanisms to prevent nitrosamine
formation from nitrate or nitrite. The most effective is to have
sufficient vitamin C present with any nitrite- or nitrate-containing
food or nutritional product. Nature has included that level of
protection in many vegetables. Most nitrate-rich vegetables
are also enriched in vitamin C. Nitrate-based products without
a specific amount of vitamin C may not confer the level of
protection needed to prevent the formation of any and all
66
nitrosamines. Perhaps the most convincing argument on the

safety of nitrite and nitrate is the fact that both are enriched
in human breast milk, nature’s most perfect food. Nursing
infants get a rich source of nitrite and nitrate, and that’s one of
the reasons that breastfed babies are typically healthier than
formula-fed infants.
However, despite all the safety data, there are two safety
concerns surrounding nitrite and nitrate. Acute toxicity is
defined by methemoglobinemia; this is sometimes referred
to as “Blue Baby Syndrome,” or cyanosis. This occurs when
nitrite causes oxidation of hemoglobin where it can no longer
carry oxygen. As a result, blueness around the lips can occur
due to lack of oxygenation. The fatal dose of nitrite is in the
range of 22 to 23 mg/kg body weight, which would translate
into about 1750 mg for a 180-pound adult. This dose is
approximately 150 times higher than doses that have been
used therapeutically in humans.
The other concern with nitrite and nitrate is the potential to

form low molecular weight N-nitrosamines, some of which
are carcinogenic. In the 1970s, there became a major public
health concern regarding nitrite exposure, either through
diet or industrial exposure, and formation of N-nitrosamines.
The first report in the 1950s on the cancer-causing effects
of N-nitrosodimethylamine (NDMA), and the suggestion that
low molecular weight N-nitrosamines can be formed following
nitrosation of various amines ignited an enormous interest
in N-nitrosamines and their association with cancer. There
are biologically plausible mechanisms that nitrite could form
nitrosamines if you created a contrived environment. NDMA
was detected in nitrite preserved fish. It was later demonstrated
that nitrosamines could form in the acidic conditions of the
human stomach. Since the early 1980s, there have been
numerous reports on the association of N-nitrosamines and
human cancers. More recent epidemiological evidence, as
well as review of a biologically plausible mechanism, has
refuted previous evidence of a causal relationship between
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NATHAN S. BRYAN
nitrite and nitrate exposure and cancer formation. Despite this

more recent evidence, there continues to be epidemiological
studies demonstrating an association between dietary nitrite,
nitrate intake, and certain forms of cancer.
As a physiologist, I’m trained to look at the big picture, and

here’s what I see. The NTP—the gold standard for assessing
safety—found that nitrite did not cause cancer in a controlled
study that was evaluated by a panel of experts. My research
and many others’, including researchers at the National
Institutes of Health, have found nitrite to be an effective
treatment for a number of health conditions without adverse
effects. Our body makes nitrite. And vegetables like spinach,
celery, cabbage, radishes, broccoli, rhubarb, and melons—
the types of foods we are encouraged to consume for good
health—are rich sources. When used to cure meat, nitrite
gives meat like salami and ham their characteristic color and
flavor and prevents the deadliest foodborne illness: botulism.
We must look at all issues in terms of a risk benefit analysis.

Nitrite and nitrate, when consumed at levels found in foods,
have important health effects and are even being considered
as essential nutrients. For those wanting more information on
this subject matter, I would recommend the book Nitrite and
Nitrate in Human Health and Disease (http://www.springer.
com/us/book/9783319461878).
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-9-
Defining Nitrite and
Nitrate as Nutrients
A nutrient is defined as any substance that nourishes an

organism, and to nourish is to sustain with food or nutriment—
to supply with what is necessary for life, health, and growth.
Some categories of nutrients include water, protein,
carbohydrates, vitamins, minerals, fatty acids, and amino
acids. There are obviously many specific examples within
each of the categories. What is clear, though, is that nutrients
are fundamental to physiological systems, and proper nutrition
can prevent many diseases. On the contrary, lack of nutrients
can cause disease. The information already presented in this
book demonstrates that nitrite and nitrate, when consumed
or administered in the “right” concentrations, under the right
conditions, can prevent or mitigate many diseases and improve
physical performance, and therefore, classify as nutrients.
Basic science and epidemiological research have shown

that all conditions of insufficient nitrite and nitrate from the
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NATHAN S. BRYAN
diet promote or accelerate disease. To the contrary, there

are many studies now showing sufficient nitrite and nitrate
from dietary sources can prevent or treat many diseases
and enhance physical fitness. As we advance the science of
nitric oxide, it is obvious from many basic science and clinical
studies that nitrite and nitrate, through the proper delivery at
the right doses, have an enormous impact on many diseases
that affect so many people today. Not dissimilar to vitamin K
metabolism (phylloquinone or vitamin K1), nitrate serves as
the primary plant form of the nutrient, whereas its metabolite
nitrite is the active form (menaquinones for vitamin K) that first
requires metabolic activation. The purpose of this chapter is
to define the context for consideration of nitrite and nitrate as
nutrients and outline optimal therapeutic levels that can easily
be achieved through diet.
Origins of nitrite and nitrate
Despite historical use of inorganic nitrate and nitrite as

medicinal agents—and the fact that these anions are
produced naturally in the body from the oxidation of nitric
oxide—along with recent demonstration of physiological roles
for nitrate and nitrite in vascular and immune function, public
perception is that these are harmful substances in our food
and water supply. That myth was dispelled in the previous
chapters. The early studies on nitrogen balance in humans
demonstrated that nitrite and nitrate are synthesized de novo
in the intestine. It was these early findings by Tannenbaum
et al. that significantly altered our thinking about human
exposure to dietary nitrite and nitrate. Prior to those studies,
it was thought that steady-state levels of nitrite and nitrate
in humans originated solely from the diet and from nitrogen-
fixing enteric bacteria.
The endogenous production of NO from the 5-electron

oxidation of L-arginine by the enzymes nitric oxide synthase
(NOS) is a fundamental physiological process that maintains
and regulates cardiovascular function, immune function and
70
neurotransmission. These discoveries were so profound

that the 1998 Nobel Prize in Physiology or Medicine was
awarded to three U.S. scientists for the discovery of NO in the
cardiovascular system. Once produced, NO has a half-life of
approximately one second, and is quickly oxidized to nitrite
and nitrate or reacts with amino acids on proteins. In fact, it has
been shown that about 50 percent of the circulating levels of
plasma nitrite reflect endogenous NO production, and steady
state levels of plasma nitrite and nitrate can be affected by
diet. Patients with compromised NO production from NOS,
termed endothelial dysfunction, have reduced levels of
plasma nitrite concentrations, and endothelial dysfunction
is associated with several cardiovascular disorders. Further
studies have shown that insufficient NO production is not only
associated with all major cardiovascular risk factors—such as
hyperlipidemia, diabetes, hypertension, smoking, and severity
of atherosclerosis—but also that it has a profound predictive
value for the future atherosclerotic disease progression.
Therefore, steady-state levels of nitrite shown to be reflective
of endogenous NO production are critical determinants of
cardiovascular disease risk and progression.
Nitrite and nitrate have been used for centuries in curing

and preserving meats and fish and in manufacturing certain
cheeses. When added to foods, specifically meats for curing,
nitrite has at least three functions. First, it contributes to the flavor
due to the inhibition of the development of rancidity. Second,
it reacts with myoglobin to give mononitrosylhemochrome,
which forms the characteristic pink color of cured meat.
Third, it inhibits the growth of food spoilage bacteria, most
importantly Clostridium botulinum. C. botulinum thrives under
anaerobic conditions and produces a neurotoxin that is one of
the most lethal natural products known. In this regard, nitrite is
critical to the food industry to prevent food borne illness from
C. botulinum.
Vegetables contribute over 85 percent of the daily dietary

intake of nitrate, In addition, endogenous synthesis is an
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NATHAN S. BRYAN
important contributor to human’s overall exposure of nitrate.

Hord et al. estimated that approximately 80 percent of dietary
nitrate is derived from vegetable consumption; therefore the
primary source of exposure to nitrate by humans is through
eating vegetables. Most people would not argue that a diet rich
in fruits and vegetables is healthy. Recent reports have shown
that less than 5 percent of the ingested nitrite and nitrate are
derived from cured meat sources, with the remainder coming
from vegetables and saliva.
Perhaps the most compelling argument for defining dietary

nitrite and nitrate requirements comes from our knowledge on
the amount of nitrite and nitrate present in breast milk of nursing
mothers and their nutritional and immunological benefits to
the infant. Previously published studies in our lab reveal the
presence of high concentrations of nitrite and nitrate in human
breast milk, consistent with previous reports. Early post-partum
breast milk from certain mothers contained the highest nitrite
concentration of any food or beverage product tested (near
20 µM, or 50 times higher than that found in beetroot juice).
At birth, the gastrointestinal tract of the infant is sterile, and
it is rapidly colonized by bacteria originating from the mother
and the environment. We now know that reduction of nitrate
to nitrite requires the commensal bacteria that normally reside
in our body. However, in newborn infants, this pathway has
not yet developed. Thus, breast milk—high in nitrite relative
to nitrate—overcomes nature’s deficiency early in life. At later
stages of development, nitrate becomes the predominant
anion when a symbiosis exists with the colonized bacteria.
This becomes extremely interesting in terms of the level of
nitrite exposure based on ingestion and body weight of an
infant (near 1 mg/kg). Comparing these values, you begin to
see discrepancy based on ignorance in terms of regulation of
nitrite and nitrate exposure.
Similar to all essential or indispensable nutrients, intake of

excess nitrate and nitrite exposure can be associated with
increased risk of negative health outcomes, specifically
72
low blood pressure or hypotension or methemoglobinemia

(blue baby syndrome). A set of Dietary Reference Intake
(DRI) categories are set by the Food and Nutrition Board of
the National Academy of Sciences for essential nutrients in
order to clearly define, where possible, the contexts in which
intakes are deficient, safe, or potentially excessive. These
DRI categories include the Recommended Dietary Allowance
(RDA), Adequate Intake (AI), Tolerable Upper Level Intake
(TUL), and Estimated Average Intake (EAI). The process
of establishing DRIs for nutrients considers a broad range
of physiological factors, not the least of which is nutritional
status and potential toxicities. Such methodologies, including
the consideration of normal dietary consumption patterns of
nitrate- and nitrite-containing foods, have not been applied
in setting exposure limits for or in considering the potential
health benefits of dietary nitrate and nitrite. There are ranges
for levels of intake that put people at risk of inadequacy and
levels that increase the risk of excess and toxicity.
So are we at risk for being exposed to toxic levels through

our diet? The National Research Council report, “The Health
Effects of Nitrate, Nitrite, and N-Nitroso Compounds” (NRC
1981), estimates nitrite and nitrate intake based on food
consumption tables. They report that the average total nitrite
and nitrate intake in the U.S. was roughly 1 mg and 76 mg,
respectively, per day. The mean intake estimates for nitrate
and nitrite in the U.S. and Europe vary by investigator, but are
consistent and comparable. International estimates of nitrate
intakes from food are 31 to 185 mg/day in Europe and in the
U.S. about 40 to 100 mg/day. The bioavailability of dietary
nitrate is 100 percent. Nitrite intakes vary from 0 to 20 mg/day
or up to 0.25 mg/kg body weight. Nitrate intakes from sources
other than vegetables, including drinking water and cured
meats, have been estimated to average 35 to 44 mg/person
per day for a 60 kg human. These average intakes are much
less than what is required to see any health benefits and 1000
times less than what would cause any toxicity.
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NATHAN S. BRYAN
Based upon a conservative recommendation to consume

400 grams of different fruits and vegetables per day at
median nitrate concentrations, dietary concentration of nitrate
would be ~157 mg/day. Assuming an average weight of the
population to be 80kg, this equates to 1.96 mg/kg per day for
nitrate. In the EU population, where fruit consumption (nitrate
concentration averaging <10 mg/kg FW) constitutes over half
the 400 gram intake recommendation, actual nitrate intakes
would approximate 81 to 106 mg/day before additional nitrate
losses are taken into account, based on washing, peeling,
and/or cooking. My lab reported that persons consuming the
DASH (Dietary Approaches to Stop Hypertension) diet could
consume upward of 1000 mg of nitrate per day, or roughly
12.5mg/kg per day.
There are however, regulations that have been issued to

prevent any toxicity. The current regulations regarding nitrite
and nitrate exposure were established based on potential
toxicology, primarily methemoglobinemia, and without
any regard for potential health benefits. The permissible
concentration of nitrate in drinking water is 50 mg nitrate
per Liter (L) in the European Union (EU) and 44 mg per L
in the U.S., in agreement with World Health Organization
recommendations first established in 1970 and reaffirmed
in 2004. The Joint Food and Agricultural Organization/World
Health Organization has set the Acceptable Daily Intake (ADI)
for nitrate at 3.7 mg/kg body weight and for the nitrite ion at
0.06 mg/kg body weight. The WHO ADI level for nitrate (0 to
3.7 mg/kg) translates into an equivalent of 222 mg nitrate for
a 60 kg adult. The fact that typical consumption patterns of
vegetables and fruit exceed regulatory limits for dietary nitrate
calls into question the rationale behind current nitrate and
nitrite regulations. The physiologic basis for regulating human
consumption of plant foods containing nitrate and nitrite should
be reevaluated to include potential health benefits.
As Paracelsus exclaimed, “Dose makes the poison.” There are

clear and defined doses of both nitrite and nitrate that provide
74
indisputable evidence of promoting health and even treating

serious medical conditions. Fortunately, these doses fall well
below toxic and fatal doses. This provides a sufficient range
for the normal dietary guidelines to be established. With such
a huge difference in therapeutic and beneficial doses for nitrite
and nitrate to those that are potentially toxic and harmful, this
provides enough room to establish nutrient recommendations
and DRIs. Until now, there has been no reason to consider
an RDA for nitrite or nitrate, which is the average daily level
of intake sufficient to meet the nutrient requirements of nearly
all (97 to 98 percent) healthy people. Tolerable Upper Intake
Level (TUL) can clearly be established, which is the maximum
daily intake unlikely to cause adverse health effects.
At a time when the world is faced with epidemics of heart

disease, obesity, and metabolic syndrome, we can no longer
ignore fundamental nutritional, biochemical, physiological,
and clinical benefits of nutrients found in the most healthy
and nutritious foods—nitrite and nitrate—especially since the
etiology of these diseases is based on poor diet and nutrition.
As with any drug or nutrient, it is time to consider the risk
benefit analysis of nitrite and nitrate. The cardiovascular
benefits are clear. The risk of exposure of nitrite and nitrate
and developing cancers is weak at best, but still important to
consider. If we consider the WHO statistics from 2013, there
were 8.2 million deaths worldwide from cancer. It is estimated
that about 30 percent of cancer deaths are due to the five
leading behavioral and dietary risks: high body mass index,
low fruit and vegetable intake, lack of physical activity, tobacco
use, and alcohol use. This would result in 2.46 million deaths
from dietary and lifestyle habits, where nitrite and nitrate per
se are not involved, but formation of N-nitrosamines may or
may not be involved in the mechanism.
Consuming more vegetables would lead to higher intakes of

nitrite and nitrate. According to WHO 2012 statistics, there
were 17.3 million deaths worldwide due to cardiovascular
disease, representing 30 percent of all global deaths.
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NATHAN S. BRYAN
Lowering blood pressure by just 5 mmHg reduces the risk

of stroke by 35 percent and risk of ischemic heart disease
by 21 percent, the top two killers of people worldwide.
There are now clear, indisputable blood pressure lowering
effects of dietary nitrite and nitrate by at least 5 mmHg. By
establishing dietary guidelines for nitrite and nitrate, perhaps
35 percent—or roughly 6 million—deaths could be prevented
each year. The ultimate question is how many, if any, new
cases of cancers would arise from consuming an amount of
nitrite and/or nitrate (that may form N-nitrosamines) to avoid
cardiovascular disease. It may be impossible to determine,
but we predict the cardiovascular benefits will far outweigh
any risk of new cancers. It should also be highlighted that
nitrite and nitrate have never been directly implicated in
carcinogenesis, but only through formation of carcinogenic
low molecular weight N-nitrosamines. Vitamin C and other
antioxidants very effectively inhibit nitrosation reactions.
As we begin to recognize safe and effective delivery systems

for nitrite and nitric oxide, we can begin to develop new
technologies that will certainly have enormous benefit to
human health. So, once thought as a harmful toxic molecule in
our food supply, nitrite is now considered an essential nutrient
and molecule produced in our body to regulate a number of
physiological functions. In fact, the emerging physiological
data on nitrite are strikingly analogous to a vitamin. We have
referred to nitrite as a vitamin previously, and even proclaimed
it “vitamin N,” but perhaps it may fit the characteristics of a
dietary mineral. A mineral is by definition a solid inorganic
substance of natural occurrence. After all, sulfates and
phosphates are recognized minerals, and nitrite and nitrate
are similar in structure and composition, except replacing the
sulfur and phosphorus with nitrogen respectively with different
oxidation states. How we classify nitrite and nitrate may not
be important at this stage other than to finally recognize them
for the nutrients they are.
76
What we have to avoid as a scientific community and food-

based companies is to begin to fortify our entire food supply
with nitrite and nitrate, such as with iron or folate. In fact,
beetroot bread was recently tested and found to lower diastolic
blood pressure and increase endothelium-independent
vasodilation. There has to be regulated and scientifically sound
technologies to deliver therapeutic or dietary supplements
containing nitrite and/or nitrate. The underlying chemistry
of these two anions must be controlled to maximize the
benefits while preventing any unwanted nitrosation chemistry
causing N-nitrosamine formation. Nutritionists, physiologists,
physicians, toxicologists, and dieticians need to converge and
establish nutrient guidelines for nitrite and nitrate similar to
other essential nutrients. The scientific evidence and facts are
now available for such an initiative. Becoming more evident, is
the enormous benefit of dietary nitrite and nitrate in a number
of disease models. A simple ubiquitous molecule we have
been advised to avoid may be an indispensable nutrient many
are lacking.
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How Much Do You Need?
It should now be clear that nitrite and nitrate from the diet can
restore loss of NO production from pathway 1, the L-arginine
pathway. There are many studies showing the health benefits
of dietary nitrate in humans, including blood pressure regulation
and sports performance. In early studies conducted in mice,
relatively high doses of nitrate protected against the damaging
effects of cancer chemotherapy by maintaining mitochondrial
function. A much lower dose of nitrate in the drinking water
of mice that cannot make nitric oxide can reverse clinical
characteristics of diabetes and metabolic syndrome. For the
majority of studies in humans, beetroot juice has been the
dietary nitrate source of choice. However, to obtain sufficient
nitrate levels for improved physical performance, a minimum
of 300 to 400 mg of nitrate needs to be provided at least 2.5
hours prior to exercise in order to allow sufficient time for the
uptake and metabolism of nitrate to nitrite and NO.
Research has shown that raising the nitrate and nitrite levels
in the body prior to exercise by consumption of dietary
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NATHAN S. BRYAN
nitrate can increase NO production and lead to increased

oxygen efficiency and exercise performance. Additionally,
in another study in older adults, dietary supplementation
with beetroot juice, containing approximately 400 mg nitrate
per dose twice per day (816 mg total), increased plasma
nitrite concentration, reduced blood pressure, and positively
influenced physiological responses to exercise. Nitrate
supplementation can also enhance exercise performance
in patients with peripheral artery disease. Improvements in
exercise performance following beetroot juice consumption
has typically been found to range between 2 to 16 percent for
different types of exercise including running, cycling, rowing,
and resistance exercise.
There are also several studies showing blood pressure

lowering effects of nitrate when consumed through beetroot
juice. Studies show that nitrate can lower blood pressure
six hours later in healthy adults. Studies out of the United
Kingdom had patients drink beetroot juice that contained
468 mg of nitrate and found that all forms of nitrate led to
a dose dependent decrease in blood pressure beginning
after ninety minutes and lasting for several hours. Similarly,
other studies demonstrate that drinking beetroot juice (442
mg nitrate per day) acutely reduces blood pressure and the
oxygen cost of submaximal exercise, and that these effects
are maintained for at least fifteen days if supplementation is
continued. Interestingly, in another study, supplementation
of the diet with nitrate enriched beetroot juice for two weeks
did not lower blood pressure, improve endothelial function, or
improve insulin sensitivity in individuals with Type II diabetes.
There appears to be patient populations that do not respond
to nitrate.
Administration of roughly 500 mg nitrate for four weeks to

older patients with increased cardiovascular risk profiles
can reverse vascular dysfunction. There is a relatively large
range of nitrate dosing that has been studied in humans and
mice that provides clear therapeutic benefit without any signs
80
of toxicity. The published studies show reductions in blood

pressure of anywhere from 2 to 10 mm Hg. This change
in blood pressure is very meaningful. A simple 5 mm Hg
reduction in blood pressure can reduce the risk of stroke by
30 percent and the risk of heart disease by 20 percent. With 2
out of 3 Americans having high blood pressure, this approach
can provide significant and profound effects on the burden of
cardiovascular disease and public health.
The question then becomes, how many vegetables do I

need to consume to get enough nitrite and nitrate in my diet
to achieve these results? The science is very clear that one
needs about 300 to 400 mg of nitrate in a single serving to
achieve the positive benefits of NO in regard to blood pressure
management and enhanced performance. Part of my research
program set out to answer that exact question. In collaboration
with the Department of Food Science at Texas A&M University,
we tested five different green vegetables from five different
cities across the U.S. We measured the nitrate content of
broccoli, cabbage, celery, spinach, and lettuce with the intent
to try to determine how many servings of each would one
need to eat to get enough nitrate to reach the 300 to 400 mg
needed. We went to New York, Raleigh, Dallas, Chicago, and
Los Angeles and gathered these vegetables from the same
retail grocer. We then took them back to the lab and analyzed
them for their nitrate content. We compared conventionally
grown vegetables to organically grown vegetables. What
we found really surprised us. There was a greater than ten
times difference in the nitrate content in specific vegetables
from one city to another. Furthermore, organically grown
vegetables had less nitrate than conventionally grown. The
amount of nitrate in each vegetable from each city is shown in
the tables below.
What this tells us is that if you lived in Dallas or Chicago, you

could eat approximately 160 grams of celery and get enough
nitrate from that celery to affect NO production. That is about
three to four celery stalks. However, if you lived in New York or
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NATHAN S. BRYAN
Chicago, you would have to eat thirty to fifty stalks of celery to

get a NO benefit. The same held true for spinach and lettuce as
well. What about organic? We are all told that organic is better,
right? Well in terms of nitrate, organically grown vegetables
contain less nitrate than conventionally grown vegetables. So,
organic may be better for you since they contain no herbicides
or pesticides, but they do not assimilate nitrogen into nitrate,
likely due to insufficient nitrogen in the soil from organic farms.
It is clear from the published clinical trials that you need 300 to
400 mg of nitrate in a single serving of vegetables to experience
the benefits discussed in the previous chapters. But so is
the realization that depending on where you live and what
type of vegetables you are eating, people may not be getting
enough nitrate from their diet. Nitrate assimilation into nitrate
is dependent upon soil conditions, time of harvest, amount of
fertilizer added, and water availability (drought). Historically,
databases have been kept in order to keep track of how much
nitrate is ingested. Based on these existing databases, the
mean estimated intake for nitrate and nitrite in the U.S. and
Europe varies but are consistent and somewhat comparable.
International estimates of nitrate intakes from food are 31 to
185 mg/day in Europe and in the U.S. about 40 to 100 mg/
day. We know from above that 300 to 400 mg in a single
serving is required for NO production and improvements in
blood pressure and exercise performance. Most people are
consuming only half of this amount over two to three meals,
and not as a single serving.
Therefore, the U.S. diet is depleted in nitrate. As a result,

Americans are a nitrate deficient society. The research
suggests that this deficiency may be partly responsible for
the increased incidence of all cardiovascular related diseases
in the U.S. population. Consistent with this notion, certain
diets provide much more nitrate. For example, the Dietary
Approaches to Stop Hypertension (DASH) diet can provide
as much as 1200 mg nitrate per day from choosing certain
foods. The Japanese diet also provides more than 500 mg
82
nitrate that has been shown to reduce blood pressure and

improve performance. Similarly, the Mediterranean diet
provides sufficient nitrate along with antioxidants to support
reduction of nitrate to nitrite and nitric oxide. There is strong
evidence that it is the dietary nitrate and nitrite in these foods
and diets that confer the protective and health promoting
activities. Although many clinical trials have been performed
to try to identify the mechanism of action of these diets, looking
primarily at antioxidants, vitamins, and minerals, most have
failed to recapitulate the effects of the whole food diets.
Evidence strongly suggests that it is the nitrate/nitrite content

along with the antioxidants that account for the effects.
However, it is not as simple as we would hope. Nitrate itself is
inactive and without effect. Nitrate must first be reduced by oral
commensal bacteria to nitrite, and then nitrite has biological
activity. As described above, only about 5 percent of the total
nitrate is reduced to nitrite. All biological effects of nitrate are
abolished by antiseptic mouthwash that kills oral bacteria.
The next chapter will reveal how to best utilize functional nitric
oxide nutrition to restore NO production.
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NATHAN S. BRYAN
84
85
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Now What? Simple Steps
to Regain your Health
The benefits of dietary nitrite and nitrate are indisputable. The

amount you need to see drastic improvements in health and
wellness are far below levels that would cause any concern
for toxicity or harmful effects. Even if we consume sufficient
nitrate from our diet, if it is not first metabolized and reduced
to nitrite, the human body cannot utilize it to make nitric oxide.
Humans do not have a functional nitrate reductase gene.
As we have learned from earlier chapters, this metabolism
is dependent upon oral bacteria. Nitrate ingested from the
diet is rapidly absorbed in the small intestine, taken up into
circulation where it mixes with the endogenous nitrate from
oxidation of nitric oxide, and readily distributed throughout the
body. About 25 percent of oral nitrate from diet is concentrated
and excreted by salivary glands, so that salivary nitrate
concentration is approximately ten times higher in the saliva
than in plasma. Approximately 20 percent of salivary nitrate
can be reduced to nitrite in the mouth by facultative anaerobic
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NATHAN S. BRYAN
bacteria, which are found on the dorsal surface of the tongue

if nitrate-reducing bacteria are present, resulting in about a 5
percent reduction of total ingested nitrate to nitrite.
If we are consuming 300 to 400 mg nitrate from our diet, which

is the dose known to be effective at reducing blood pressure
and enhancing performance, then this reduction efficacy
results in 15 to 20 mg nitrite produced from dietary nitrate
through the enterosalivary circuit. Nitrate, when consumed
through the diet, reaches peak blood levels in about an hour.
The levels will remain elevated for about five to six hours.
The high concentration of nitrate and nitrite in saliva and other
tissue, continuous production from nitric oxide, and the re-
absorption from renal tubules strongly suggest that nitrate
and nitrite have a definite role in normal human physiology
and are not just unwanted toxins. They serve as important
substrates for NO production, provided the body can utilize
them.
So how can you optimize your nitric oxide production? As

mentioned earlier, the evidence suggests that the U.S. diet is
deficient in nitrate. Furthermore, common drug therapy and
lifestyle decisions disrupt metabolism of nitrate into nitrite
and nitric oxide. So, people taking antibiotics, antiseptic
mouthwash, and/or antacid drugs further disrupt the benefits
of getting nitrate from the diet. How does one overcome
deficiencies in dietary nitrate, variability between individual
microbiomes, mouthwash use, PPI use, or insufficient
stomach acid production? We and others have focused on
nitrite. Nitrite is the metabolic product of nitrate reduction by
the bacteria. Nitrite itself can be utilized by human enzyme
systems to generate and produce NO, and is not dependent
upon oral bacteria. Nitrite is derived directly from exogenous
dietary nitrate, but also from the oxidation of endogenously
produced nitric oxide. Nitrite is found naturally in colostrum
and breast milk, small amounts found naturally in green
leafy vegetables, and small amounts added to cured meats.
88
Whereas daily nitrate intakes vary from 150 to 300 mg from

the diet, nitrite intake from food varies from 5 to 40 mg/day.
When we measure actual NO production in young healthy

adults, this varies between 0.15 and 2.2 μmol/kg/hour. When
we convert this production rate to the amount of nitrite this
produces as a consequence of sufficient NO production, for a
150- to 170-pound person this would equate to approximately
20 to 200 mg nitrite and nitrate daily. Due to approximately 5
percent reduction of nitrate (average of 150 mg per day in U.S.
diet plus 200 mg from oxidation of NO), this would equate to
17.5 mg endogenous nitrite production. Therefore, total daily
nitrite exposure in a normal healthy individual on a Western
diet is roughly 20 to 40 mg. For the same healthy individual
consuming more of a vegetarian diet or DASH diet that
included 400 to 1200 mg nitrate per day, endogenous nitrite
production could exceed 70 mg per day. These nitrite levels are
dramatically reduced in people with endothelial dysfunction,
insufficient vegetable consumption, or consuming vegetables
without sufficient nitrate along with use of antibiotics/antiseptic
mouthwash and/or PPIs.
These data beg the question that if most people are nitrite
deficient, can we safely and adequately supplement back
what is missing? This approach is no different than vitamin D,
for example. If labs demonstrate we are low in vitamin D, then
you supplement what is missing in order to normalize your
levels. This has been our approach with nitrite and nitrate.
There are a number of published studies in humans showing

the safety and efficacy of nitrite within a large range of doses.
Sodium nitrite capsules at doses of 160 mg and 320 mg were
used to determine toxicity and pharmacokinetics. Nitrite even
at a dose of 320 mg did not show any clinically toxic levels of
methemoglobinemia (<15 percent). However, some subjects
reported mild headache and nausea that resolved after a half-
hour. This study also revealed that nitrite is 98 percent orally
available. Another study using a sodium nitrite capsule in
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NATHAN S. BRYAN
diabetics demonstrated that a single administration of 80 mg

sodium nitrite was well tolerated with no significant changes
in any measures of toxicity. The 80 mg nitrite dose led to a
significant drop in systolic blood pressure with no effect on
diastolic pressures.
Chronic studies using 80 to 160 mg nitrite capsules for ten

weeks in a randomized, placebo-control, double-blind study
increased plasma nitrite acutely and chronically and was well
tolerated without an unsafe drop in blood pressure. Endothelial
function, measured by brachial artery flow-mediated dilation,
was significantly improved without changes in body mass
or blood lipids. Carotid artery elasticity (as measured by
ultrasound and applanation tonometry) improved. These
functional changes were related to eleven specific metabolites
that could predict the vascular changes with nitrite. Similarly,
in another study using 80 and 160 mg nitrite capsules for ten
weeks showed improvement in performance on measures of
motor and cognitive outcomes in healthy middle aged and
older adults (62 ± 7 years). These studies provide evidence
that sodium nitrite supplementation is well tolerated, increases
plasma nitrite concentrations, improves endothelial function,
lessens carotid artery stiffening, and improves motor and
cognitive function in middle-aged and older adults, perhaps
by altering multiple metabolic pathways. The effects of nitrite
are not dependent upon oral nitrate-reducing bacteria, and
appear to be safe even at doses that far exceed daily human
production.
Sodium nitrite also appears to have a positive effect in

compromised patients. In a double-blind, randomized,
placebo-controlled, parallel-group trial, subjects with heart
failure with preserved ejection fraction (HFpEF) underwent
invasive cardiac catheterization with simultaneous expired
gas analysis at rest and during exercise, before and fifteen
minutes after treatment with either sodium nitrite or a matching
placebo. Before the nitrite infusion, HFpEF subjects displayed
an increase in pulmonary capillary wedge pressure (PCWP)
90
with exercise from 16 ± 5 mm Hg to 30 ± 7 mm Hg (p < 0.0001).

After nitrite infusion, the primary endpoint of exercise PCWP
was substantially improved by nitrite compared with the
placebo (adjusted mean: 19 ± 5 mm Hg vs. 28 ± 6 mm Hg; p
= 0.0003). Nitrite-enhanced cardiac output reserve improved
with exercise and normalized the increase in cardiac output
relative to oxygen consumption. Nitrite improved pulmonary
artery pressure-flow relationships in HFpEF and increased
left ventricular stroke work with exercise versus placebo,
indicating an improvement in ventricular performance with
stress (Borlaug et al. 2015). These authors conclude that acute
sodium nitrite infusion favorably attenuates hemodynamic
derangements of cardiac failure that develop during exercise
in individuals with HFpEF.
The doses of nitrite used in these studies are typically more

than one would normally consume in an ordinary diet. This is
in part due to the fact that nitrite is inefficiently reduced to NO
along the physiological oxygen gradient, and therefore more
is needed to get any appreciable amount of NO produced—
especially in people who are NO deficient. Through the
discovery of natural product chemistry of an oxygen
independent nitrite reductase, lower supplemental doses of
nitrite can more effectively reduce nitrite to NO and therefore
provide an exogenous source of NO in the oral cavity. The
premise of this technology is that if your body can’t make
NO due to endothelial dysfunction, oral dysbiosis, antiseptic
mouthwash, or PPI use, then this will provide an exogenous
source of NO.
Studies using a patented composition of matter formulation

(Neo40™, HumanN, Inc™)—with a standardized amount
of nitrite along with a functional nitrite reductase for the
generation of NO to account for differences in endogenous
production along with the natural product chemistry in the
form of an orally disintegrating tablet—found that nitrite
could modify cardiovascular risk factors in patients over
the age of 40, significantly reduce triglycerides, and reduce
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NATHAN S. BRYAN
blood pressure. Single administration of this lozenge leads

to peak plasma levels of nitrite around 1.5 µM. In patients
with argininosuccinic aciduria (ASA), the nitrite lozenge led
to a significant reduction in blood pressure when prescription
medications were ineffective, improved renal function and
cognition, and reversed cardiac hypertrophy. Another
randomized controlled study using the nitrite lozenge showed
that a single lozenge can significantly reduce blood pressure,
dilate blood vessels, and improve endothelial function and
arterial compliance in hypertensive patients. Furthermore, in
a study of pre-hypertensive patients (BP >120/80 < 139/89),
administration of one lozenge twice daily leads to a significant
reduction in blood pressure (12 mmHg systolic and 6 mmHg
diastolic) after thirty days, along with improvements in
functional capacity as measured by a six-minute walk test. In
an exercise study, the nitrite lozenge significantly improved
exercise performance.
Most recently, in subjects with stable carotid plaque, the NO

lozenge led to a 11 percent reduction in carotid plaque after
six months. To put this in perspective, meta-analysis of trials
using treatment with statins (cholesterol-lowering medication)
reported that a total of seven trials showed regression
and four trials showed slowing of progression of CIMT of
approximately 2.7 percent (-0.04) after more than two years.
Using the nitric oxide lozenge, the data show an average of
0.073 mm or 10.9 percent after six months. Similarly, this
same patented technology in the form of a concentrated
beetroot powder (Superbeets™, HumanN, Inc.™) attenuates
peripheral chemoreflex sensitivity without concomitant change
in spontaneous cardiovagal baroreflex sensitivity, while also
reducing systemic blood pressure and mean arterial blood
pressure in older adults. These studies clearly demonstrate
the safety and efficacy of low supplemental doses of nitrite in
humans that can correct for any insufficiencies from dietary
exposure, pharmacological inhibition by antiseptics, or PPIs.
92
Is this what nature intended?
If supplementing deficient nitrite and nitrate in populations is a

viable strategy for combatting cardiovascular disease or any
condition associated with insufficient nitric oxide availability,
are there examples in the epidemiology literature with specific
populations that can provide justification for such? The answer
may come in the form of nature’s most perfect food, breast
milk. We and others have published analyses showing that
early breast milk and colostrum contain high concentrations
of nitrite until the bacteria begin to colonize in the oral and
digestive tract of growing infants. Once commensal bacteria
have colonized, breast milk changes from nitrite to nitrate so
that the infant’s body can utilize the nitrate-reducing bacteria
to provide a more extended exposure to nitrite. Commercial
infant formulas lack any nitrite and have very little nitrate. The
health disparities between breastfed and formula-fed babies
are well known. Supplementing nitrite that is missing in formula
can protect from necrotizing enterocolitis.
Additionally, anthropological studies on native Tibetans reveal

that their acclimatization to living at high altitude and reduced
oxygen is through increased nitric oxide production with 20 to
50 times higher circulating nitrite and nitrate than those who
live at sea level. People who live at or near sea level increase
their NO production and plasma levels of nitrite and nitrate as
they ascend to altitude. Increasing nitrite and nitrate availability
is a physiological response to low oxygen, and the adaptive
response to allow us to acclimate to different environments. In
other words, increasing steady state concentrations of nitrite
and nitrate appears to be a natural physiological response that
allows the body to adapt to changing oxygen environments,
whether environmental or physiological.
To summarize, there are really some simple steps you can take
to ensure sufficient and adequate nitric oxide production to
maintain healthy circulation, blood flow, and cellular function.
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NATHAN S. BRYAN
If you follow the steps below you can allow your body to make
NO and be in the best health you can be.
1. Eat more green leafy vegetables.
2. Get moderate physical exercise (twenty minutes, three to

five times per week).
3. Stop using antiseptic mouthwash and do not overuse

antibiotics.
4. Stop using antacids. You need stomach acid to make NO.
5. Supplement your diet with standardized nitric oxide

functional nutrition products.
The next question is, how do you know what standardized

nitric oxide functional nutrition products are? There are many
nitric oxide products on the market. When searching for a
“functional” nitric oxide product, look for four things:
1. Are there any published clinical trials conducted on the

actual product? Clinical trials are expensive to conduct,
and most dietary supplements are not rigorously studied
in clinical trials.
2. Are there any patents listed on the product label? Patents or

licenses from universities or medical schools demonstrate
that the product is innovative and unlike any other product
on the market.
3. Is the product from a reputable company that is focused

on nitric oxide innovations? There are many reputable
companies that make good products that aren’t related
to nitric oxide. NO is a complicated science and requires
expertise and know how. If the company isn’t experienced
in NO, then their products will typically not work.
94
4. Has the inventor or formulator of the product ever published

in the nitric oxide field? Many people can put ingredients
together, but one must be experienced in nitric oxide
research. Peer-reviewed publications are a great metric
for determining if the company or formulator is an expert
in nitric oxide.
These are simple steps to make sure you find and take
products that work. Functional nitric oxide nutrition may be the
most important consideration for your health and wellness.
The published science tells us that your body cannot and will
not heal or perform optimally without fixing the dysfunction
nitric oxide production pathways. I trust the information in this
book will provide you with the information and knowledge to
help you make decisions that will improve your health.
95
- 12 -
Looking Forward
Regular intake of nitrate- and nitrite-containing foods may

ensure that blood and tissue levels of nitrite and NO pools
are maintained at a level sufficient to compensate for any
disturbances in endogenous NO synthesis from NOS. Since
low levels of supplemental nitrite and nitrate have been shown
to enhance blood flow, dietary sources of NO metabolites can,
therefore, improve blood flow and oxygen delivery, and protect
against various cardiovascular disease states or any condition
associated with NO insufficiency. As science advances and
new discoveries and understandings are made, it is important
to be able to incorporate these new findings into meaningful
guidelines that can enhance health, lengthen life, and reduce
illness and disability. Translating new science discoveries to
medical practice or public health policy takes approximately
seventeen years, which is way too long for the health benefits
to be realized.
We now know that “Dose makes the poison.” Fortunately the

doses of both nitrite and nitrate that are effective at lowering
97
NATHAN S. BRYAN
blood pressure, improving circulation and performance are

about 100 times less than what would cause any toxicity.
Furthermore, the safe and efficacious doses can be obtained
from foods and standardized supplements. Amounts found
naturally in food will never reach levels of toxicity. The
science has shown clearly that a healthy diet should not only
focus on reducing sugar and caloric intake, but on adding
foodstuffs promoting nitric oxide bioactivity, which can include
foods enriched in nitrite, nitrate, L-arginine, and antioxidants
to promote NO production and availability. This pathway
is also the current focus of a number of biotechnology and
pharmaceutical companies in their attempts to develop NO-
and nitrite-based therapeutics.
In 2016, total healthcare expenditures in the U.S. exceeded

$3.3 trillion up 4% from the previous year, or $10,348 per
person, accounting for almost 18% of the Gross Domestic
Product (GDP). This trend is expected to increase over the
coming years, increasing by 5.6% every year for the next ten
years. Currently, costs associated with chronic diseases such
as obesity, diabetes, hypertension, coronary artery disease
account for 75 percent of the nation’s annual healthcare costs.
According to the American Heart Association, an estimated
92 million people had one or more forms of cardiovascular
disease in the U.S. in 2016, (up 12% from a 2006) including
hypertension, coronary artery disease, myocardial infarction,
angina pectoris, stroke, and heart failure. By 2030, 43.9% of
the US adult population is projected to have some form of CVD.
Most, if not all, chronic diseases that lead to the highly prevalent
burden of cardiovascular disorders, including diabetes and
obesity, are the result of a dysfunctional endothelium and
inability to produce NO and/or maintain NO homeostasis and
signaling. Understanding and developing new strategies to
restore NO homeostasis will have a profound impact on public
health and on the health care system. Defining the context for
the role of nitrite and nitrate in human health and disease is an
essential first step in the process.
98
We are at the beginning of a new field in nutritional biochemistry

as it relates to nitrite and nitrate. Historically, we have relied
on databases for average nitrite and nitrate content of
certain and specifics foods. Then, based on food frequency
questionnaires, we are able to assess a daily exposure value
for both nitrite and nitrate. Our most recent survey of both
vegetables and cured and processed meats report very large
differences in nitrite and nitrate content of certain meats and
vegetables, but also regional differences as well as differences
between conventional versus organically grown vegetables. In
fact, there exists as much as a thirty-fold difference between
conventional nitrate content of conventionally grown celery
between cities, and as much as a fifty-four-fold difference
in organically grown broccoli between cities. This presents
obvious problems with using food frequency questionnaires
and dated databases for estimations of nitrite and nitrate
exposure. The variation in compositional content, specifically
nitrate concentration, of organically and conventionally
produced raw vegetables (highly consumed) may need to be
considered when compiling nutrient composition databases.
If compositional differences are of sufficient magnitude, this
might warrant an “organic” category in databases to be an
additional factor to consider when modeling nutrient intake.
For epidemiological studies investigating nitrite and nitrate
exposure, it will be necessary to obtain and measure the actual
foods consumed in order to get an accurate value for individual
exposures. Furthermore, long-term controlled studies need to
be conducted to establish set amounts of nitrite and/or nitrate
consumption to determine if certain doses can actually have
an effect on the onset and/or progression of chronic disease
in humans. The only way to do this is through functional nitric
oxide nutrition with standardized amounts of nitrite and nitrate
in foods and supplements.
The most reasonable conclusion that can be made from the data
reviewed is that humans are adapted to receive dietary nitrite
and nitrate from birth and throughout life, and therefore nitrite
99
NATHAN S. BRYAN
and nitrate may not pose significant risks at levels naturally

found in certain foods. In fact, we believe the absence of
these essential nutrients in our diets may be involved in many
of the chronic health problems facing the entire developing
world. Advancements in science and research over the past
thirty years have illuminated the essential nature of nitrite and
nitrate in our food supply as well as how our body makes these
natural molecules. Eating a well-balanced, nutritious diet and
performing moderate exercise comprise the ideal model of
routinely good health and disease prevention. The role of a
proper diet in the prevention of disease is well established
by many population-based epidemiological studies. Nothing
affects our health more than what we choose to eat.
Nitric oxide is essential for maintaining normal blood pressure,

preventing adhesion of blood cells to the endothelium, and
preventing platelet aggregation; it may, therefore, be argued
that this single abnormality, the inability to generate NO, puts
us at risk for diseases that plague us later in life. Developing
strategies and new technologies designed to restore NO
availability is essential for inhibiting the progression of these
common chronic diseases. The provision of dietary nitrate and
nitrite may allow for such a strategy. In fact, understanding
how the body utilizes key dietary nutrients, specifically nitrate,
will help scientists and physicians develop more effective
treatment strategies for overcoming key limitations in our diet
or metabolism of dietary constituents. Indeed, implementing
functional nitric oxide nutrition has the potential to profoundly
change the face of health and disease.
100
About the Author
Dr. Bryan earned his undergraduate Bachelor of Science

degree in Biochemistry from the University of Texas at Austin,
and his doctoral degree from Louisiana State University
School of Medicine in Shreveport, where he was the recipient
of the Dean’s Award for Excellence in Research. He pursued
his post-doctoral training as a Kirschstein Fellow at Boston
University School of Medicine in the Whitaker Cardiovascular
Institute. After a two-year post-doctoral fellowship, in 2006 Dr.
Bryan was recruited to join faculty at the University of Texas
Health Science Center at Houston by Ferid Murad, M.D.,
Ph.D., 1998 Nobel Laureate in Medicine or Physiology.
During his tenure as faculty and independent investigator at

UT, his research focused on drug discovery through screening
natural product libraries for active compounds. His nine
years at UT led to several discoveries, which have resulted
in more than a dozen issued US and international patents
and many more pending. Specifically, Dr. Bryan was the
first to describe nitrite and nitrate as indispensable nutrients
required for optimal cardiovascular health. He was the first
to demonstrate and discover an endocrine function of nitric
oxide via the formation of S-nitrosoglutathione and inorganic
nitrite. Through the drug discovery program in natural product
chemistry, Dr. Bryan discovered unique compositions of
matter that can be used to safely and effectively generate
and restore nitric oxide in humans. This technology is now
validated in multiple published clinical trials.
Dr. Bryan is also a successful entrepreneur who has

commercialized his nitric oxide technology through the
formation and Founding of a University of Texas Health
Science Center portfolio company, named to the Inc 5000
fastest growing companies in the US for many years ongoing.
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NATHAN S. BRYAN
Dr. Bryan has been involved in nitric oxide research for

several decades and has made many seminal discoveries in
the field. These discoveries and findings have transformed
the development of safe and effective functional bioactive
natural products in the treatment and prevention of human
disease and may provide the basis for new preventive or
therapeutic strategies in many chronic diseases. Dr. Bryan
has published a number of highly cited papers and authored
or edited 5 books. He is an international leader in molecular
medicine and nitric oxide biochemistry. He is also a rancher
raising registered Hereford cattle. In his free time, Dr. Bryan
enjoys golfing and roping.
102
About Dr. Bryan’s Work
Dr. Bryan has spent his entire professional career researching

nitric oxide. His research program has always been very
focused on two main objectives:
1. To understand what goes wrong in people who can’t make

nitric oxide.
2. Identify nutritional and therapeutic strategies to fix the

underlying biochemistry and physiology to restore normal
NO production.
This endeavor has resulted in discoveries that have the

potential to change the management and prevention of
chronic disease. Loss of nitric oxide is the earliest event
in the onset and progression of most, if not all, chronic
diseases. Understanding how to fix the underlying problem
of NO insufficiency will transform public health and disease
management.
His current research projects include identification and

characterization of oral nitrate reducing bacteria that provide
the human host with a continuous source of nitrite and
nitric oxide. Lack of these oral bacteria leads to chronic NO
deficiency and all that entails, including increase in blood
pressure, exercise intolerance, poor circulation, and loss of
nitric oxide-based signaling. This new paradigm then focuses
on the oral microbiome as a therapeutic target to manage
blood pressure and nitric oxide-based functions.
Restoring nitric oxide production in the human body is essential

for humans to perform to their full potential and prevent age-
related disease. Your body cannot and will not heal until you
fix the nitric oxide production pathways.
103
Other Books by Dr. Bryan
Beet the Odds: Harness the Power of Beets to Radically

Transform Your Health by Nathan S. Bryan, PhD, Carolyn
Pierini, CLS (ASCP), CNC – Neogenis Laboratories 2013
Food, Nutrition and the Nitric Oxide Pathway: Biochemistry and

Bioactivity by Nathan S. Bryan, PhD - DesTech Publications,
Inc. 2010
Nitrite and Nitrate in Human Health and Disease by Nathan S.

Bryan, Joseph Loscalzo – Humana Press 2011
The Nitric Oxide (NO) Solution: How to Boost the Body’s

Miracle Molecule to Prevent and Reverse Chronic Disease by
Nathan S. Bryan, PhD, Janet Zand, OMD, with Bill Gottlieb,
CHC – Neogenis Labs 2010
105
Connect with the Author
Website: www.drnathansbryan.com
Email: drnathanbryan@gmail.com
Social Media:
Facebook:
https://www.facebook.com/nathan.bryan.16
LinkedIn:
https://www.linkedin.com/in/nathan-bryan-27586b7/
Twitter: @drnathanbryan
107
Acknowledgements
I have had many mentors, teachers, and colleagues that

have inspired me to ask the tough questions, design good
experiments, and recognize all scientific observations are
meaningful even if it was not what was expected. These
include my first mentor, Martin Feelisch, Ph.D, good friend and
colleague Tienush Rassaf, M.D., Ph.D., friend and co-author
on a previous book Joseph Loscalzo, M.D., Ph.D., and Ferid
Murad, M.D., Ph.D., who hired me for my first faculty position.
David Lefer, Ph.D., who mentored me and collaborated with
me as I established my research program. John P. Cooke,
M.D., Ph.D., who is someone I have always looked up to
and respected in the NO field. C. Thomas Caskey, M.D., for
believing in my science and encouragement early on in my
commercial ventures. Last but certainly not least, my family
for their unending support and encouragement even during
my many nights away from home.
109
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Resources
www.drnathansbryan.com
www.functionalnitricoxidenutrition.com
www.nitricoxidesociety.org
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Functional Nitric Oxide Nutrition:

Dietary Strategies to Prevent and Treat Chronic Disease

Copyright © 2018 by Nathan S. Bryan

Crescendo Publishing, LLC

ISBN: 978-1-948719-00-1 (p)

Printed in the United States of America

Click on the link below the image to hear a special message

Dr. Nathan Bryan and I have been educating doctors at the

As a women’s health specialist, I speak on the critically

Dr. Bryan is a true trailblazer in this still infant field of nitric

I am an integrative gynecologist and teach doctors around the

I celebrate the research Nathan has done in the field of nitric

Felice Gersh, M.D.

2. What is Nitric Oxide and How is it Produced?.......................... 17

3. How is Nitric Oxide Derived from Nutrients?............................. 27

4. Bacteria: Helping Us Do What We Cannot Do......................... 35

5. Focus on Nitrogen-Based Nutrients........................................... 45

6. Why Vegetables are Good for You............................................. 49

7. Undisputed Health Benefits......................................................... 55

8. But I Thought Nitrite and Nitrate Were Toxic............................ 63

9. Defining Nitrite and Nitrate as Nutrients.................................... 69

10. How Much Do You Need?........................................................... 79

11. Now What? Simple Steps to Regain your Health..................... 87

12. Looking Forward............................................................................ 97

About the Author................................................................................ 101

About Dr. Bryan’s Work.................................................................... 103

Other Books by Dr. Bryan................................................................ 105

Connect with the Author................................................................... 107

~ Dr. Dennis Burkett

Numerous blood tests and noninvasive cardiovascular

later pathway is most important in patients after the age of 40

This new data has huge implications to developing treatment

Dr. Bryan has performed a masterpiece of literary genius in his

Mark C. Houston MD MS MSc FACP FAHA FASH FACN

Health care and medicine have been and continue to be

However, the development of safe and effective treatments

These statistics suggest that although people are living longer,

The intentions of this book are to highlight new science that

lifestyle modifications can significantly improve nitric oxide

1. Nitric oxide is one of the most important molecules

2. Loss of nitric oxide is the earliest event in the onset and

3. As we age, we lose the ability to produce nitric oxide,

4. New discoveries in science and medicine reveal what

With these four basic concepts and by employing the strategies

This book is organized to provide a historical account of

that are necessary and sufficient for adequate NO production.

Next, I describe why vegetables are good for you. It goes

The field of nitric oxide is relatively new. The molecule was

Advancements in nutrition and discoveries of new nutrients

disease, the number one killer of men and women worldwide.

Today more than ever, gaining nutrition knowledge can

In 400 B.C., Hippocrates, the “Father of Medicine,” said to his

In the same era as Hippocrates in 400 B.C., foods were often

references to food and health. One story describes the

During the 1500s, scientist and artist Leonardo da Vinci

Lind didn’t know it at the time, but he had discovered a vital

In 1790, George Fordyce recognized calcium as necessary for

the natives developed a disease called beriberi, which caused

In the early 1900s, E.V. McCollum, while working for

Crescendo Publishing, LLC 