Dietary Nitrite and Nitrate: From Menace to Marvel

Nathan S. Bryan, Ph.D.

Institute of Molecular Medicine

Reciprocal Meat Conference

Lubbock, TX June 20-23, 2010

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 later—that is,

from 2006 to 2009—the agency

approved 74. Not among them were

any cures, or even meaningfully

effective treatments, for Alzheimer’s

disease, lung or pancreatic cancer,

Parkinson’s disease, Huntington’s

disease, or a host of other afflictions

that destroy lives.

Newsweek, May 15, 2010

Current Environment

Time and Money

Discovery to FDA Approved Drug – 10 years and $800,000,000

“It takes physicians an average of 17 years to adopt widely the

findings from new basic research.”

U.S. Senator and thoracic surgeon, Dr. William Frist

2005 Shattuck Lecture published in the New England

Journal of Medicine

Functional foods are "those foods that encompass potentially healthful

products including any modified food or ingredient that may provide a

health benefit beyond the traditional nutrients it contains," as defined by

the Institute of Medicine. Functional foods can include foods like cereals,

breads and beverages which are fortified with vitamins, herbs and

nutraceuticals.

The healing power of foods is a popular concept that focuses on how

"super foods" can have health protecting properties. Medicinal foods or

"nutritionally high powered foods" have been part and parcel of the natural

products industry for a long time and, through emerging scientific research

and particularly through growing public interest, they have reached the

mainstream.

Functional Foods

Established Benefits of a Single Molecule

Protect from heart attack and stroke (Bryan et al PNAS 2005,

Shiva et al JEM 2007)

Enhances exercise performance (Bailey et al & Larsen et al)

Restores NO biochemistry in eNOS knockout mice (Bryan et al FRBM 2006)

Reverses hypertension from NOS inhibition (Tsuchiya et al AJP 2005)

Inhibits microvascular inflammation, reversing endothelial dysfunction

and reduces levels of C-reactive protein (Stokes et al AJP 2009)

Plays a role in host defense (Lundberg et al Gut 1994)

Maintains gastric mucosal integrity (Benjamin et al Nature 1994)

Prevents Ulcerative Colitis (Ohtake et al Nitric Oxide 2010)

Inhibits Cancer Cell Proliferation (Morcos E et al Nutr Cancer. 2010)

What is this Hero Molecule?

Nitrite

Structure of Presentation

Review on Nitric Oxide

Current Paradigm on NO Production/Regulation

Nitrogen Cycle: Nature’s NO

Disease Intervention in mice and Humans

Current perspectives

Disclosure: N.S. Bryan is Chief Science Officer and has a financial interest

in NeoGenis Labs, Inc. (www.neogenis.com)

What is Nitric Oxide?

The chemical compound nitric oxide is a gas with chemical formula NO٠.

It is an important signaling molecule in the body of mammals including humans,

one of the few gaseous signaling molecules known.

It is also a toxic air pollutant produced by automobile engines and power plants.

NO should not be confused with nitrous oxide (N2O), a general anesthetic, or with

nitrogen dioxide(NO2) which is another poisonous air pollutant.

The nitric oxide molecule is a free radical, which is relevant to understanding its

high reactivity. It reacts with the oxygen in air to form nitrogen dioxide, signaled

by the appearance of the reddish-brown color.

NO

Cardiovascular System

Respiratory Tract

Immunology

Cell Proliferation

Central Nervous System

Peripheral

Nervous System

Gastrointestinal/

Urogenital Tract

Vasorelaxation

Blood Cell Regulation

Myocardial Contractility

Microvascular Permeability

Bronchodilatation

Asthma, ARDS

NANC nerve-mediated

Relaxation

Learning and Memory

Pain Sensitization

Epilepsy

Neurodegeneration

Central BP Control

Apoptosis

Angiogenesis

Tumor Cell Growth

Unspecific Immunity

Inhibition of Viral Replication

Transplant Rejection

Penile Erection

Pre-term Labour

Nitric Oxide Plays a Key Role in the Regulation of

Numerous Vital Biological Functions

Nitroglycerine, a 150 year old explosive and heart medicine

In atherosclerosis, plaques

reduce blood flow in the

arteries. This decreases oxygen

supply to the heart muscle

causing chest pain (angina

pectoris) and sometimes even

myocardial infarction.

Treatment with nitroglycerine

provides NO, dilates the

vessels, and increases blood

flow.

Thanks to the 1998 Nobel

Laureates we now understand

how nitroglycerine, an important

heart medicine, works.

It acts as a NO donor, causes

dilation of the blood vessels,

increases oxygen supply

and protects the heart from

damage and cell death.

Reduced NO availability is a hallmark of a

number of cardiovascular disorders.

- Endothelial dysfunction is a physiological dysfunction of normal biochemical

processes carried out by the endothelium, the cells that line the inner surface

of all blood vessels including arteries and veins (as well as the innermost lining

of the heart and lymphatics).

- Loss of endothelial NO function is associated with several cardiovascular disorders,

including atherosclerosis, which is due either to decreased production or to increased

degradation of NO (Davignon and Ganz 2004).

- Experimental and clinical studies provide evidence that defects of endothelial NO function,

referred to as endothelial dysfunction, is not only associated with all major cardiovascular

risk factors, such as hyperlipidemia, diabetes, hypertension, smoking and severity of

atherosclerosis, but also has a profound predictive value for the future atherosclerotic disease

progression (Schachinger, Britten et al. 2000; Halcox, Schenke et al. 2002; Bugiardini,

Manfrini et al. 2004; Lerman and Zeiher 2005).

- The dysfunctional eNOS/NO pathway is considered as an early marker or a common

mechanism for various cardiovascular disorders. Over the last two decades, it has become

evident that decreased bioavailability of endothelial NO, produced from endothelial NO

synthase (eNOS), plays a crucial role in the development and progression of atherosclerosis.

10 20 30 40 50 60 70

0

20

40

60

80

100

% D

eclin

e in

NO

Pro

du

ctio

n

Age in years

men

women

Gerhard et al Hypertension 1996

Celermajer et al JACC 1994

Taddei et al Hypertension 2001

Egashira et al Circulation 1993

Similar to HGH, Humans lose their ability

to produce NO as we age

L-Arg

Diet

L-Arg

ArginaseADMA

Transport

NOSUncouplingReduced OxygenReduced Cofactor + SubstrateOxidative Stress

Antioxidants

NO2

NO3

Oxidation

Bacterial Reduction

NO

+

O2-٠

ONOO-

Health

Disease

BH4 Ca/Cam

FAD+ FMN

NADPH O2

Heme iron GSH

Mitochondria

XO

NADPH oxidase

The L-Arginine-Nitric Oxide Pathway

Urea Cycle

L-Arg

Current Paradigm

Despite almost 30 years of research after its discovery, very few clinical drugs on the market for NO.

- organic nitrates (used for over 150 years for treatment of angina)

- inhalative NO therapy for neonates

- phosphodiesterase inhibitors such as sildenafil (Viagra) which act downstream from NO

L-Arginine supplementation is ineffective and in fact detrimental in some populations

L-arginine therapy in acute myocardial infarction: the Vascular Interaction With Age in Myocardial

Infarction (VINTAGE MI) randomized clinical trial. (JAMA. 2006 Jan 4;295(1):58-64)

CONCLUSIONS: L-arginine, when added to standard postinfarction therapies, does not improve vascular

stiffness measurements or ejection fraction and may be associated with higher postinfarction mortality. L-

arginine should not be recommended following acute myocardial infarction.

L-Arginine Paradox

Km for NOS is 5µM. Plasma levels of L-arginine ~100µM

How can you get modest improvement in NO output when enzyme theoretically saturated with

substrate?

Physiological systems are fraught with redundancy. Where is the redundant

NO pathway? Why does such a critical molecule only have a singular complex

and complicated pathway to production????

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

Atmospheric Nitrogen CycleThe store of nitrogen found in the atmosphere, where it exists as a gas (mainly N2), plays an important role for life. Most

plants can only take up nitrogen in two solid forms: ammonium ion (NH4+ ) and the nitrate ion (NO3- ). Most plants obtain

the nitrogen they need as nitrate from the soil. When released, most of the ammonium is often chemically altered by a

specific type of bacteria (genus Nitrosomonas) into nitrite (NO2- ). Further modification by another type of bacteria (genus

Nitrobacter) converts the nitrite to nitrate. All nitrogen obtained by animals can be traced back to the eating of plants at

some stage of the food chain.

New Paradigm - Human Nitrogen Cycle

Dietary nitrate and nitrite intake

Commensal bacteria reduce 20% of nitrate to nitrite

Low gastric pH reduces nitrite to NO

Systemic circulation –nitrite and nitrate delivered throughout the body

25% circulating Nitrate concentrated in salivary glands

Nitrate and nitrite absorbed by intestines

Majority of Nitrate Filtered and reabsorbed

Mice on Low NOx Diet for 1 Week Reveal

Diminished Plasma and Cardiac Nitrite and Nitrate

and is Restored with Supplemental Nitrite

Bryan et al PNAS (2007)

Mice on Low NOx Diet for 1 Week Reveal

Increase Injury from Heart Attack which

is Reversed with Supplemental Nitrite

Bryan et al PNAS (2007)

Circulating and Tissue Nitrite/Nitrate are

Affected By Both NOS and Diet and

Restored by Supplemental Nitrite

*

*

*

Plasma Heart

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Nitri

te [

µM

]

C57 Std Diet

eNOSKO Std Diet

eNOSKOLowNOx

+ Nitrite

*

Plasma Heart

0

5

10

15

20

25

30

35

40

Nitra

te [µ

M]

C57StdDiet

eNOSKOStdDiet

eNOSKOlowNOx

+ Nitrite

Bryan et al, FRBM 2008

Supplemental Nitrite Reverses MIR Injury

in eNOS -/- mice

Bryan et al, FRBM 2008

LDL

LDLEndothelium

Vessel Lumen

Intima

Monocyte

Modified LDL

Macrophage

T Cells Neutrophils

Smooth MusclesProliferation

Atherogenesis

Foam cells

LESION

Hypothesis: Dietary nitrite acts as an alternate source of NO and provides anti-inflammatory

properties to inhibit leukocyte adhesion to endothelium.

Nitrite in food controls and stabilizes the oxidative state of lipids in meat products

(Shahidi and Hong 1991), thus preventing lipid oxidation. However nitrite is known as a

strong oxidant in human physiology by its propensity to form methemoglobinemia.

Emerging physiological evidence reveal that nitrite may have anti-inflammatory properties

as well akin to NO. The goal is to determine if supplemental nitrite can inhibit leukocyte

rolling and adhesion in a mouse model of vascular inflammation.

Group I - Controls: C57 mice fed normal diet for 3 weeks with nitrite free MQ water ad libitum

Group II – HC diet: C57 mice fed high cholesterol diet for 3 week with MQ water ad libitum

Group III – low nitrite: C57 mice fed high fat diet for 3 weeks with 50mg/L nitrite added to

drinking water ad libitum

Group IV – high nitrite: C57 mice fed high fat diet for 3 weeks with 150mg/L nitrite

The 50mg/L dose of nitrite has been shown to be cardioprotective in I/R injury and inhibit plaque

formation in atherosclerotic mice without producing any measurable changes in methemoglobin.

______________________________________________________________________________

Groups Cholesterol Triglyceride MAP WSR Leukocyte Count

(mg/dL) (mg/dL) (mmHg) (s-1) (#/mL blood)

__________________________________________________________________________________

ND+water 71±2.8 57.0±27 64±3.3 733±66.8 4830±990.9

HC+water 116±4.2* 75.3±14.05 67±0.7 594±27.0 6767±1238.2

HC+50mg/L nitrite 117±8.9* 47.3±5.68** 69±1.2 708±65.8 6590±785.7

HC+150 mg/L nitrite 123±7.7* ND 69±2.7 756±44.8 6080±761.3

__________________________________________________________________________________

* P<0.005 vs. ND+H2O

** P<0.01 vs. ND+H2O

ND – not determined

Nitrite Supplementation Reduces

Triglycerides in Hypercholesterolemic Mice

Stokes, et al. AJP 2009

High Fat Diet

High Fat Diet + Nitrite

High Fat Diet Induces Microvascular Inflammation that

Is inhibited by nitrite supplementation

# Adherent Leukocytes # Emigrated Leukocytes

0

200

400

600

800

1000

1200

1400

1600

1800

2000

**

#

*

# c

ells

/mm

2 v

esse

l w

all

Normal diet + water

HC diet + water

HC diet + 50 mg/L nitrite

HC diet + 150 mg/L nitrite

#

*

Plasma Heart Liver

0.0

0.5

1.0

1.5

2.0

2.5

80

120

160

200

*

Nitri

te [µ

M]

Normal diet + water

HC diet + water

HC diet + 50 mg/L nitrite

*

#

Stokes, et al. AJP 2009

High Fat Diet Causes Endothelial Dysfunction that

Is reversed by Nitrite Supplementation

ACh dilation

0

10

20

30

40

50

60

70

80

90

100

110

120

**

% R

ela

xa

tio

n

Normal Diet + water

HC diet + water

HC diet + 50 mg/L nitrite

HC diet + 150 mg/L nitrite

#

Control HC diet HC diet + nitrite

0

50

100

150

200

250

300 #*

BH

4/B

H2

ra

tio

Stokes, et al. AJP 2009

High Fat Diet Causes Increase in C-reactive Protein.

Nitrite Lowers CRP Induced by High Fat Diet

Control HC

HC low nitrite

HC high nitrite

0

10

20

30

40

50

CR

P (

ng

/ml)

p = 0.02

Stokes, et al. AJP 2009

Nitrite Nitrate

0

5

10

15

20

NO

x [

µM

]

BreastMilk

Formula

Nitrite in Breast Milk is Absent in

Formula

0.25mg/kg

0 2 4 6 8 10 12 14 16 18

0

5

10

15

20

25

30

35

40

NO

x [

µM

]

Days post-partum

Nitrite

Nitrate

Nitrite is Enriched in Colostrum and

Declines As Gut Bacteria Colonize

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 NO-

related effects in cardiovascular field. However, their mechanism of action is

far from clear.

Traditional Chinese Medicines

TABLE 2: Quantitative assessment of nitrite, nitrate, nitroso, nitrite reductase activity and the recommended daily dose for each.

Data represented are an average of n=3-4 ± SEM for nitrite and nitrate, nitroso and nitrite reductase (each measurement was with

fresh TCM on separate days).

Nitrite Nitrate Nitroso

Anaerobic NO

production

Aerobic NO

production daily dose

(µg/g) (µg/g) (nmoles/g) (pmole/mg) (pmole/mg)

DS 0.33 ± 0.06 11948 ± 2192 745 ± 418 6.99 ± 4.21 1.4 ± 0.3 5-10g/day

XB 0.26 ± 0.05 278 ± 76 146 ± 48 45.0 ± 12.3 46.0 ± 19.3 5-9g/day

GL 0.15 ± 0.03 530 ± 110 996 ± 108 133.9 ± 31 66.4 ± 56.4 3-9g/day

SC 0.21 ± 0.01 2069 ± 127 449 ± 33 12.6 ± 1.8 44.2 ± 28.2 3-10g/day

RX 0.98 ± 0.59 61 ± 6 195 ± 124 71.5 ± 20 190.6 ± 100 3-10g/day

CS 0.12 ± 0.03 37 ± 1.5 21.2 ± 15.0 255 ± 177 19.2 ± 9.0 3-10g/day

HS 0.30 ± 0.10 243 ± 30 64.4 ± 20 359 ± 104 255 ± 55 5g/day

TCMs Reduce Nitrite to NO

0 1000 2000 3000 4000 5000 6000

0

20

40

60

80

100

120N

O [p

pb

]

Time (min)

DSJ

DS

XBGL

SQRX

CS

HS

SBP

NBP

TCMs generate NO from Nitrite

Tang et al FRBM 2009

Herbs Generate NO and Relax

Blood Vessels and Reverse Endothelial Dysfunction

1E-3 0.01 0.1 1 10

0

20

40

60

80

100

120

###

#

#

#

#

****

*

% R

ela

xa

tio

n

Acetylcholine [µM]

Control

ApoEKO

ApoEKO+TCM

*

What about Functional Foods?

Nitrite (µg/g) Nitrate (µg/g)

Nature’s Way Garden Veggies 8.9 12053

Beet <1 10383

Artichoke <1 13243

Spinach 50 58

Green Tea <1 74

Pine Bark <1 2319

Tribulus 3.9 8276

0 1000 2000 3000 4000 5000

0

20

40

60

80

100

120

NO

[p

pb

]

Time (seconds)

B

Kinetics of NO Formation after

Rational Combination of Herbs

NO3-

NO2-

NO

Manipulating the System

Through Natural Product Chemistry

oxidation reduction

probiotics

Herbs with reductive

capacity

Beet, artichoke, etc

Oxygen,

ceruloplasmin

Oxyheme

proteins

L-arginine

Small Trial in Humans Using Unique

Combination of Herbs with Highest NO

ActivityPatients enrolled in Austin Tx

Licensed chiropractor, acupuncturist

30 day regimen bid

Patient 1

55 yom

Non-smoker

Plasma nitrite

Day 0 Day 30

0.23 0.11

Alanine Aminotransferase (ALT)

Day 0 Day 30

18.5 11.1

C-Reactive Protein (CRP)

Day 0 Day 30

4.03 0.36

Total Triglycerides

Day 0 Day 30

128 91

Total Cholesterol

Day 0 Day 30

138 146

Patient 2

52 yof

smoker

Plasma nitrite

Day 0 Day 30

0.13 0.09

Alanine Aminotransferase (ALT)

Day 0 Day 30

9.3 3.7

C-Reactive Protein (CRP)

Day 0 Day 30

0.52 0.21

Total Triglycerides

Day 0 Day 30

209 135

Total Cholesterol

Day 0 Day 30

168 148

Patient 3

46 yof

smoker

Plasma nitrite

Day 0 Day 30

0.10 0.10

Alanine Aminotransferase (ALT)

Day 0 Day 30

3.7 5.8

C-Reactive Protein (CRP)

Day 0 Day 30

2.29 0.67

Total Triglycerides

Day 0 Day 30

177 137

Total Cholesterol

Day 0 Day 30

139 133

Nitrite Has Dose Dependent Effects

In Different Stages of Colon Cancer

01nM

10nM100nM

1µM10µM

100µM

0

10000

20000

30000

40000

50000

*******

* *

Flu

ore

sce

nce

In

ten

sity

Nitrite Concentration

SW1116

SW480

HCT15

COLO205

A

01µM

10µM100µM

250µM500µM

1000µM

0

10000

20000

30000

40000

50000

*

*

**

*

*

*F

luo

resce

nce

In

ten

sity

Ascorbic Acid Concentration

SW1116

SW480

HCT15

COLO205

B

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

Nitrate – Nitrite – NO pathway may be a redundant pathway for maintenance

Of NO homeostasis to compliment the L-arginine pathway

Utilizing activity from natural products and products that contain nitrite and

nitrate can provide an essential source of bioactive NO

Over 3000 years of phase I safety data

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 vascular disease.

"Vitamine" or vital amine was the term coined by Casimir Funk (1884-1967)

for the unidentified substances present in food which could prevent the

diseases scurvy, beriberi, and pellagra.

Definition of Vitamin:

- any of a group of organic substance essential in small quantities to normal

metabolism

- found in minute amounts in natural foodstuffs or sometimes produced synthetically

- deficiencies of vitamins produce specific disorders.

√√

√

At the Very Least, Nitrite and Nitrate Should be Viewed

as Indispensable Nutrients

Could it be then that if one does not eat sufficient foods rich in nitrite and nitrate

specific disorders occur? Our animal studies reveal that nitrite insufficiency

exacerbates I/R injury and a host of cardiovascular diseases are associated with

decreased NO availability as measured by nitrite. Becoming more evident is the

enormous benefit of exogenous dietary nitrite and nitrate in a number of disease

models. What is the Risk Benefit Analysis?

Nitrite acts as a Vitamin

Acknowledgements

Brown Foundation Institute of Molecular Medicine

The University of Texas Health Sciences Center at Houston

Harsha Garg

Yaoping Tang, M.D.

Hong Jiang, Ph.D.

Yong-Jian Geng, M.D., Ph.D.

LSU Health Sciences Center at

Shreveport

Karen Stokes, Ph.D.

Tammy Dugas, Ph.D.

D. Neil Granger, Ph.D.

Albert Einstein College of Medicine

David Lefer, Ph.D.

John Elrod, Ph.D.

Mark Duranski

John Calvert, Ph.D.

Susheel Gundewar M.D.

Food, Nutrition and the

Nitric Oxide Pathway:

Biochemistry and Bioactivity

Editor: Nathan S. Bryan

www.destechpub.com

ISBN: 978-1-932078-84-8,

September 2009

238 pages, 6x9, hardcover

Nitrite and Nitrate in Human Health and

Disease

Editors: Nathan S. Bryan, Ph.D.

Joseph Loscalzo, M.D., Ph.D.

Springer-Humana Press

Nutrition Series

In print Jan 2011

Fourth International Meeting on the Role of Nitrite on

Physiology, Pathophysiology and Therapeutics

May 11-13 2011, Emory University, Atlanta GA

www.nitrite2011.com