Nitric Oxide: The Miracle Molecule

Nathan S. Bryan, Ph.D.

Chief Science Officer for Neogenis Labs Professor of Molecular Medicine at the University of Texas Health Sciences Center at the Houston Institute for Molecular Medicine

Leading nitric oxide research. Changing lives.

Disclosure: N.S. Bryan is a paid consultant and Chief Science Officer of Neogenis Labs, Inc.
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Nitric Oxide: Research

One of the Most Studied Molecules In Medical History

Leading nitric oxide research. Changing lives.

Monday, June 20, 2011

What is Nitric Oxide?

A gas with chemical formula NO A free radical, reacting with the oxygen in air to form nitrogen dioxide One of the few signaling molecules in the body of mammals including humans
Naturally produced in the endothelium Literally tells surrounding muscles to relax Enhances blood flow through proper dilation

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Thousands of Trials & Published Studies

Nitric Oxide Molecule of the Year

Over 120K Articles On Nitric Oxide

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Nitric Oxide: In the body

The discovery of Nitric Oxide and its function is one of the most important in the history of cardiovascular medicine.
The American Heart Association Quoted in The New York Times
Leading nitric oxide research. Changing lives.

Monday, June 20, 2011

Cell Proliferation
Apoptosis Angiogenesis Tumor cell growth

Cardiovascular System
Vasorelaxation Blood cell regulation Myocardial contractility Microvascular permeability

Immunology
Unspecified immunity Inhibition of viral replication Transplant rejection Nitric Oxide

Respiratory Tract
Bronchodilation Asthma, ARDS

Peripheral Nervous System

NANC nerve-mediated relaxation

Central Nervous System

Learning/memory Pain sensitization Epilepsy Neurodegeneration Central BP control

Gastrointestinal/ Urogenital Tract

Penile erection Pre-term labor

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Nitroglycerine, the Original Nitric Oxide Donor

Smooth muscle Endothelium

Adventia

Plaque

Atherosclerosis=plaques reduce blood flow

decreases oxygen supply to the heart causes angina pectoris or myocardial infarction

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Nitroglycerine, the Original Nitric Oxide Donor

Smooth muscle Endothelium

Adventia

Plaque

Nitroglycerine provides NO to the narrow atherosclerotic vessel

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Cardiovascular Effects of NO

Thrombocytes

Monocytes

Granulocytes
Regulation of vascular tone and blood pressure Inhibition of migration and proliferation of smooth muscle cells

Shear stress

Inhibition of aggregation of thrombocytes and of adhesion of thrombocytes, monocytes and granulocytes at the endothelium

> anti-atherosclerotic properties

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Nitric Oxide: Disease Development

NO plays an important role in the protection against the onset and progression of cardiovascular disease.
Leading nitric oxide research. Changing lives.

2005, PubMed

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Endothelial Dysfunction is the Enemy

Sufficient Nitric Oxide levels =

endothelial cells communicate with surrounding tissues telling them to relax

Insufficient Nitric Oxide levels =

endothelial dysfunction

Endothelial Dysfunction =
contributor to all major CV diseases

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Plasma NO & Endothelial Dysfunction

Plasma NO concentrations reflect the degree of endothelial dysfunction in humans.

RISK FACTORS Hyperlipidemia Arterial hypertension Smoking Age (45 males: 55 females)

Kleinbongard et al FRBM 2006

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Nitric Oxide Production & Aging

Adults over 40 make far less Nitric Oxide than their bodies need

Gerhard et al Hypertension 1996 Celermajer et al JACC 1994 Taddei et al Hypertension 2001 Egashira et al Circulation 1993

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The Complex L-arginine Pathway

Health
Diet

Oxidation Antioxidants
Urea Cycle

L-Arg L-Arg Arginase ADMA

L-Arg

BH4 Ca/Cam FAD+ FMN NADPH O2 Heme iron GSH

NO
Mitochondria XO NADPH oxidase

NO2

NO3

Bacterial Reduction

NOS
Uncoupling Reduced Oxygen Reduced Cofactor + Substrate Oxidative Stress

O2-

Disease
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ONOO15

The Current Environment

How the road from promising scientific breakthrough to real-world remedy has become all but a dead end. From 1996 to 1999, the U.S. food and Drug Administration approved 157 new drugs. In the comparable period a decade laterthat is, from 2006 to 2009the agency approved 74. Not among them were any cures, or even meaningfully effective treatments, for Alzheimers disease, lung or pancreatic cancer, Parkinsons disease, Huntingtons disease, or a host of other afflictions that destroy lives. Newsweek, May 15, 2010
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Ways to enhance NO availability

Co-factor or Substrate Supplementation L-Arginine Ascorbic Acid Folic Acid Tetrahydrobiopterin (BH4) Nitrosothiols Nitrite/Nitrate Nitro-fatty acids Nitroglycerin/organic nitrates NO hybrid drugs (NO-NSAIDS)

Require Functional NOS System

NOS-Independent Sources of NO

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Human Nitrogen Cycle

Dietary nitrate is rapidly absorbed into the bloodstream, where it mixes with endogenous nitrate from the NOS/NO pathway. A large portion of nitrate is taken up by the salivary glands, secreted with saliva and reduced to nitrite by symbiotic bacteria in the oral cavity. Salivary-derived nitrite is further reduced to NO and other biologically active nitrogen oxides in the acidic stomach. Remaining nitrite is rapidly absorbed and accumulates in tissues, where it serves to regulate cellular functions via reduction to NO or possibly by direct reactions with protein and lipids. NO and nitrite are ultimately oxidized to nitrate, which again enters the enterosalivary circulation or is excreted in urine.

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Comparing mice on a low nitrite diet and mice receiving supplemental nitrite

Bryan et al PNAS (2007)

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Heart attack injury is reversed

Bryan et al PNAS (2007) Monday, June 20, 2011 20

Steady state levels of NOx affected both By diet and NOS

* *

Bryan et al, FRBM 2008 Monday, June 20, 2011 21

Nitrite Reverses MIR injury in eNOS-/- mice

Bryan et al, FRBM 2008 Monday, June 20, 2011 22

Atherogenesis
Monocyte T Cells Neutrophils

Vessel Lumen
LDL

LDL
LESION

Endothelium

Intima

Modified LDL

Foam cells
Smooth Muscles Proliferation
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Macrophage

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High fat diet induces microvascular inflammation that is inhibited by nitrite supplementation

Stokes, et al. AJP 2009

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Nitrite Pre-Clinical Trial High Fat Diet

Actual artery with high fat diet & endothelial dysfunction

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Nitrite Pre-Clinical TrialDiet + Nitrite High Fat

Same artery after nitrite treatment, improved blood flow

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High fat diet causes endothelial dysfunction and reversed by supplemental nitrite

Stokes, et al. AJP 2009

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High fat diet causes increase in CRP and reversed by nitrite

Stokes, et al. AJP 2009 Monday, June 20, 2011 28

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Nitrite in Breast Milk is Absent in Formula

0.25mg/kg

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Nitrite is Enriched in Colostrum and Declines as Gut Bacteria Colonize

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Traditional Chinese Medicine

Traditional Chinese Medicine (TCM) has been used as a main stream of medical care throughout Asia for centuries. But it is still considered an alternative medical system in the western world. Mostly because of a lack of understanding of their mechanisms of action and/or the active compounds. Many cardiovascular diseases are characterized by a NO insufficiency. There are a number of published reports on the association of TCM and NOrelated effects in cardiovascular field. However, their mechanism of action is far from clear.

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Traditional Chinese Medicines for CVD

Tang et al FRBM 2009

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Kinetics of NO Formation From Herbs

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Endothelial Impact of Select Herbs

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Using Natural Product Chemistry

Beet, artichoke, etc

NO3
Oxyheme proteins

Probiotics

NO2
Oxygen, ceruloplasmin

Herbs with reductive capacity

OXIDATION
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NO
L-arginine

REDUCTION
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Nitric Oxide: Restoration

Leading nitric oxide research. Changing lives.

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Study: Can Nitric Oxide be Naturally Restored?

Tibetans live at 4200 meter above sea level. The low barometric pressure causes lower arterial oxygen content and subjects the highlanders to chronic hypoxia.
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Results: Increased Blood Flow

Erzurum et al. PNAS (2007) Monday, June 20, 2011 39

Because of increased NO production

Erzurum et al. PNAS (2007) Monday, June 20, 2011 40

Nitric Oxide: Restoration

Leading nitric oxide research. Changing lives.

Monday, June 20, 2011

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Nitric Oxide: Restoration

Leading nitric oxide research. Changing lives.

Over 40

L-arginine cannot be converted

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Naturally Restoring Nitric Oxide Production

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Neo40 Daily Clinical Trial Results

Strong & sustained Nitric Oxide activity

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Neo40 Daily Clinical Trial Results

30-day regimen increased plasma nitrite and nitrate in patients

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Neo40 Daily Clinical Trial Results

Significant reduction in patients with elevated triglycerides

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Neo40 Daily Clinical Trial Results

Modest reduction in blood pressure

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Dosing & Protocol

Objective Prevention, improved absorption of dietary nutrients & supplements Therapeutic, history of CV disease or current risk factors

Dose 1 lozenge per day, dissolve on tongue 1 lozenge, 2X per day, dissolve on tongue

Routine Early in the morning on empty stomach Early in the morning on empty stomach, before dinner on empty stomach. At least 2 hours before bed.

For exercise enhancement

1 lozenge 15-30 minutes before exercise

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The First & Only Saliva Test for NO

Simple to use Instant, easy-to-read results

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Testing Protocol

Baseline Repeat every week 1 test on empty stomach

Neo40 Daily 1 lozenge on empty stomach

Test 1 15 minutes later

Test 2 2-3 hours later

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Neo40 Daily Benefits

Benefit Energy Sexual function Sleep Workout pre & post Mental acuity Absorption of nutrients Blood flow to extremities
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How it works Circulation Circulation Circulation Circulation Circulation Circulation Circulation

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Chapter 3 NOtrition: NO-Boosting Foods and Supplements Go For the Greens Theres a scientific revolution in progress, and its happening on your plate.

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NO From Plants & Botanicals

Rich source of nitrate (beet root, artichokes, etc) Contain the active nitrate and nitrite reductase, antioxidants and polyphenols that can generate nitrite and NO
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Daily NO Intake Varies By Diet

Breakfast

Western Menu
Bagel w/ Cream Cheese Black Coffee (12 oz) Carrot Nut Muffin Diet Coke (12 oz) Big Mac

Mediterranean Menu
Toast w/ Jam and Butter Cappuccino (Espresso+Milk) Yogurt Carrot Juice (12 Mediterranean Garden Vegetable Soup Mineral Water (12 Trail Mix Orange Juice (12 Salmon Red Wine (12 oz)

AM Snack
(Strawberry) oz)

Lunch
Wrap oz)

Large French Fries Diet Coke (12 oz) Snickers Black Coffee (12 oz) Cheese Pizza (4 slices) Diet Coke (12 oz)

PM Snack
oz)

Dinner
(Smoked)

H. Garg, Masters Thesis Monday, June 20, 2011 55

The NO Content of Beverages

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The Ultimate Nitric Oxide Index

Kale Swiss Chard Arugula Spinach Chicory Wild Radish Bok Choy Collard Greens Beets Chinese Cabbage Le>uce Cabbage Mustard greens Cauliower, Raw Parsley Kohlrabi Carrot Broccoli
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6825 2055 1452 1123 938 814 775 697 632 499 388 312 226 167 150 136 127 122
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Conclusions

Current paradigm for NO production through L-arginine is complex and inefficient, especially in the aging population Strategies to restore NO production/homeostasis will have a profound impact on public health Utilizing activity from natural products can overcome our inherent inefficiencies and provide an essential source of bioactive NO Rational design of NO rich herbs may provide a natural, cost effective and over the counter product for conditions related to NO insufficiency to improve health, performance and prevent disease. Recognizing foods rich in NO potential may provide information into the health benefits of certain foods/diets (Nitric Oxide Index)

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Acknowledgements

The University of Texas Health Sciences Center at Houston Harsha Garg Yaoping Tang, M.D. Ferid Murad, M.D., Ph.D. Pamela Berens, M.D.

Albert Einstein College of Medicine David Lefer, Ph.D. John Elrod, Ph.D. Mark Duranski John Calvert, Ph.D. Susheel Gundewar M.D.

LSU Health Sciences Center at Shreveport Karen Stokes, Ph.D. Tammy Dugas, Ph.D. D. Neil Granger, Ph.D.

Cleveland Clinic & Case Western Cynthia Beall S.C. Erzurum Dennis Steuhr J. Tejero

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www.destechpub.com ISBN: 978-1-932078-84-8, September 2009 238 pages, 6x9, hardcover Price: $149.50

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Book Highlights: Restoring nitric oxide production in the body thereby combating:
High blood pressure Heart attack Stroke Diabetes Arthritis Kidney disease Memory loss Osteoporosis

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