Nox magazine

VOLUME 3 • ISSUE 10

ADVANCESIN ORTHOMOLECULAR RESEARCH

NOx321 TechnologyThe Power of Nitric Oxide

research-driven botanical integrative orthomolecular innovative

The Power of NOx: Say Yes to No!

Nitric Oxide and Cardiovascular Health

Nitric Oxide, Exercise and Endurance

Nitric Oxide and Gastrointestinal Health

Antimicrobial and Healing Effects of NO

The Safety of Nitrates

2 ADVANCES in orthomolecular research Volume 3 Issue 8


Published in Canada by

Advanced OrthomolecularResearch Inc.

Publisher/Editor-in-Chief

Megan Tracey, MSc

Research & WritingTraj Nibber, BSc. MSc. Ph.DDirector of Research

Graphic Design/Art ProductionNeil [email protected] David [email protected]

Digital version of this magazine and back issues are available online at www.AOR.ca

VOLUME 3 • ISSUE 10


Advances in Orthomolecular Researchis published and distributed through integrative physicians, health carepractitioners, and progressive health food retailers.

The content of this magazine is provided for informational purposesonly, and is not intended as medical advice for individuals, which canonly be provided by a healthcare professional. Contents and design ©2010 AOR. Any reproduction in whole or part and in print or electronicform without express permission is strictly forbidden. Permission toreproduce selected material may be granted by contacting the publisher.

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1 16 21

8 18 24

1. The Power of NOx: Say Yes to No!8. Nitric Oxide and Cardiovascular Health16. Nitric Oxide, Exercise and Endurance18. Nitric Oxide and Gastrointestinal Health21. Antimicrobial and Healing Effects of NO24. The Safety of Nitrates

The Power ofNitric Oxide:Say Yes to NO!What is Nitric Oxide?Nitric Oxide (NO) is a simple molecule consisting ofone atom of nitrogen and one of oxygen, making iteven simpler than water itself! Research into the effectsof this small but important molecule began in the1970’s when scientists started to examine why bloodvessels relaxed when certain compounds were added.This led to the discovery of NO and it’s amazingeffects in the body, with NO being named themolecule of the year in 1992! In 1998, twenty-someyears after this research first began, a Nobel Prize wasawarded to these researchers for their breakthroughdiscoveries regarding NO. Unfortunately, one of thepioneering researchers in the field of NO-research,Salvador Moncada, was overlooked by the Nobel Prizecommittee, showing that there is indeed a political sideto science! Over the last two decades NO research hascontinued to grow, and there has been an exponentialincrease in the number of publications on thisfascinating molecule.

NO is now known to be a signaling molecule;meaning that is acts as a messenger to facilitatecommunication between cells, the relaying ofmessages and other cellular chatter. The body relieson a wide variety of signaling molecules includinghormones, nerve impulses and chemicals likecytokines, hydrogen and carbon dioxide. Thesesignaling molecules help the body perform vitalfunctions including the mobilization of the body’simmune defense systems against invading foreignerslike viruses, bacteria, toxins and carcinogens, as wellas the modulation of other body functions liketemperature and blood pressure.

Besides being a simple molecule consisting of two ofthe most abundant and well know elements, NO isalso a radical. This means that NO has an unpairedelectron, making it a very reactive molecule! NO’sunpaired electron means that it has extensive reactivitywith virtually anything and everything from body cellsand tissues to other chemicals and moleculescirculating in the system. This reactivity is essential inenabling NO to carry out its many biological functions.

How is NO generated?The conventional method of NO synthesis is from theamino acid L-Arginine (see Figure 1). L-Arginine isoxidized via a series of steps involving a family ofenzymes called nitric oxide synthases (NOSs). Thereare essentially three common NOSs termed isozymes;these are iNOS, eNOS and nNOS. Each of theseenzymes plays a different role in the generation of NOin different tissues like the nerves, endothelium orblood vessels or on demand. In each case however,normal oxygen conditions (also called “normoxia”) aswell as a neutral to high (alkaline) pH level are

Volume 3 Issue 10 ADVANCES in orthomolecular research 1

L-Arginine

Nitric Oxide Synthases (NOS’s)

Nitric Oxide

The Power ofNitric Oxide:Say Yes to NO!

Figure 1: The production of Nitric Oxide ( NO) fromL-Arginine by enzymes called NO synthases (NOSs)


KEY TERMS AND DEFINITIONSMediterranean Diet

OxidationDASH Diet

Bioavailability

ReductionHypoxia

Mole

Genetic Knock-Out

Free Radicals

NOS

Reactive Oxygen Species (ROS)

Consumption of fruits and vegetables has been known for some time to protect against Cardiovascular Disease (CVD) and Cancer. In the mid 1960’san ambitious study called the Seven Countries Epidemiological Study was conducted. The study included the following countries: United States,

Italy, Greece, Japan, Holland, the former Yugoslavia and Finland. A five year follow up of their diet, including intake of fruits, vegetables, saturatedfats and meat was recorded as well as the incidence of cardiovascular disease and cancer. The study was unique in that it standardized for a number offactors, including risk factors of heart disease, accurate measurement of diet and blinding (meaning that no one would know who was on which diet).

A careful and extensive analysis of the data revealed that the incidence of CVD was significantly lower in Italy, Japan and Greece than in the otherfour countries included in the study. Dr. Ancel Keys, the lead author, coined the term “The Mediterranean Diet” to describe the diet followed by the

Japanese, Italians and the Greeks. In each of these countries the intake of fruits and vegetables was significantly greater and correspondedto a reduced rate of cardiovascular disease. The Mediterranean diet is now universally recognized as a healthy diet.

(Dietary Attempts to Stop Hypertension) ismodeled after the Mediterranean diet.In the DASH diet an intake of fruits and vegetablesof upwards of 8-10 servings per day is recommended.

Oxidation is a term that means an increased number of oxygen atoms(strictly speaking it refers to the loss of electrons) for example when NO undergoes

oxidation the number of oxygen atoms increases: NO NO2 NO3.

Reduction is a term that is theopposite of oxidation and refersto the loss of oxygen atoms(again strictly speaking it is the gain of electrons)for example when NO undergoesreduction the number of oxygenatoms decreases: NO3 NO2 NO.

Bioavailability is a term that refers to the amount of an active moleculethat reaches the target site of action. Poor bioavailability is a hugeproblem for large number of natural and pharmaceutical compounds.There is active research into various ways of delivering more of the moleculeat lower doses.

Half-life is the time taken for the concentration of a compound to be reduced by half. Various metabolic processes in the bodyact to ensure that molecules do not “linger” about for longer than is necessary by degrading them via enzymatic function,

binding with various proteins, elimination via increased excretion by the kidneys, and other mechanisms.

Half Life

Hypoxia is a condition when oxygen availability is greatly reduced.For example, such a condition is likely to occur during ischemia,

which is when there is an interruption of blood flow to the tissues(e.g. the heart during a heart attack).

NOSs are Nitric Oxide Synthases,which are a diverse range of enzymes

that facilitate the formation of NOfrom various starting molecules (substrates)

like L-Arginine.

Genetic knock-out mice/rats are widely used to testwhat happens when a certain gene is deleted. When agene is deleted the mouse can no longer produce a certain proteinor enzyme. For example, using rodents lacking a specific gene thatcodes for NOS enzymes, one can test the effect of a lack of NO andits health implications.

Mole is a measurement widely used in chemistry that equates howmuch of the active molecule is present. The mole and/or milli-mole(mM - a thousandth of a mole) is widely used to express the concentrationof an active compound in the blood.

Free Radicals are molecules that are very reactive dueto an unpaired electron. Free radicals are generallyconsidered to have negative health implications but

that isn’t the universal case. For example,NO itself is a free radical!

Reactive oxygen species (ROS) is another termfor free radicals; species that are very reactive.


required. When these conditions are met the NOS-dependent conversion of L-Arginine occurs efficiently.Production of NO is the primary reason for the dietaryintake of L-Arginine. However, under low oxygenconditions (also called “hypoxia”) the conversion of L-Arginine to NO is severely limited. Low oxygenconditions can occur for a variety of reasons. Forexample, partial or complete blockage of a bloodvessel (ischemia), conditions of extreme physicalexercise or high altitude situations like mountaineeringcan all result in reduced blood flow and thus reducedoxygen delivery to the body’s tissues and cells.Moreover, such low oxygen conditions are alsoaccompanied by an increased production of lactic acidwhich reduces pH making the tissue condition bothhypoxic and acidic. As such, a body builder pumpingiron or an energetic aerobic class or a middle to longdistance runner derive little if any benefit from L-Arginine in terms of NO generation.

A New Discovery! A Second Pathway to NOGeneration: The NOx 3,2,1 pathwayHowever, there is good news for these athletes!Recently a novel pathway to NO generation fromdietary nitrates has been discovered by researchers atthe Karolinska Institute in Stockholm, Sweden and byresearchers from the University of London, England.The Swedish researchers, led by Jon Lundberg andEddie Weitzberg, along with Nigel Benjamin fromLondon, were trying to discover why certain diets, likethe Mediterranean diet, vegetarian diets, Japanesediets and the famed DASH diet (Dietary Advice forStopping Hypertension) were particularly protective ofthe heart. Both groups independently reported that thekey to the success of these diets was the consumptionof leafy green vegetables, and that a key componentof all these diets was the high nitrate content. Theresearchers proposed that the nitrate was convertedinto NO via a reductive process as follows:Essentially, the highly nitrate-rich vegetables are

reduced (see key terms pg 2) in the mouth by bacteriathat are normally present on the back of the tongue.These specialized bacteria use the nitrate to help themmake energy in the form of ATP. In return, the bacteriautilize their own nitrate reducing enzyme called NitrateReductase to generate nitrite, which as we shall see isvery useful to us! This special relationship is aninteresting example of human-bacteria symbiosis: amutually beneficial relationship.

The nitrite is a much more active molecule than nitrateand is present in high concentrations in the salivawhich is swallowed into the stomach where conditionsof low oxygen (relative to the mouth) and high acid arepresent. These conditions are ideal for furtherreduction of nitrite into NO. The entire reductionprocess of nitrate into nitrite and then into NO occurswithout the intervention of NOS enzymes. Theseenzymes wouldn’t be active in these low oxygen andlow pH conditions anyway. It should be noted that lowoxygen and low pH conditions don’t just occur in thestomach, they can also occur throughout the body incertain situations including extreme physical exercise,heart disease and psychological and physical stress!Both pathways of NO generation are depicted inFigure 3, below.

Figure 3. There are two ways that the body generates NO depicted as YingYang. Each pathway has special requirements. NO can be produced fromL-Arginine and oxygen by a family of enzymes, the NO synthases (NOSs).In a more recently discovered pathway, inorganic nitrate and nitrite arereduced to form NO in blood and tissues when oxygen levels are low.Although NO generation by the L-Arginine pathway is ineffective as oxygenlevels fall, the nitrate–nitrite–NO pathway is enhanced! (From Lundberg etal., 2008)

Nitrate (NO ) Nitrite (NO ) Nitric Oxide (NO)3 2- -

Conversion of nitrate tonitrite by bacterial enzymesin the mouth

Reduction of nitrite toNO in the stomachor blood

L-Arginine Nitric Oxide(NO)

Nitric Oxide(NO)

Nitrate & Nitrite(NO /NO )

NormalOxygen Conditions(Normoxia)

LowOxygen Conditions(Hypoxia)

NOS-DependantPathway

NOS-IndependentPathway

Oxidation Reduction

3 2- -

Figure 2: The conversion of dietary nitratesto nitrite and finally to nitric oxide.


Oxidation is a step-wise increase in the number ofoxygen and reduction is the opposite with oxygenbeing reduced from 3 to 2 to 1; hence the termNOx3,2,1 pathway. Besides specific conditions likeoxygen status and pH, other factors like the presenceof certain reducing compounds will also help toenhance the conversion of nitrates into NO. Thesehelpful reducing compounds include vitamin C, alphalipoic acid and many polyphenols like green teacatechins (EGCG) and resveratrol.

Once NO is generated in the stomach it is rapidlyabsorbed across the cell membrane into the bloodvessels and from there it is carried all over the body.However, as mentioned before, NO is a free radicaland is therefore a highly reactive species that lasts onlya few milliseconds (a few thousandths of a second)before it is oxidized back into both nitrite and nitrates.

See Table 1 for a detailed comparison between nitrate,nitrite and NO.

All of this begs the question: “How is it that a moleculewith such a short half-life can exert biological effects at

Nitrate in the blood can also bepicked up by the salivary glandsand start the cycle of NOproduction all over again

Some nitrate and nitrite reach theblood and can be convertedinto NO when oxygen levels are low

Excess nitrate is excretedby the kidneys

Any left over nitrate and nitrite areabsorbed in the intestine

The acidic environment of the stomachresults in the conversion of nitrite toNO. This NO can go on to have effectsthroughout the body

Bacteria in theoral cavity convertnitrate to nitrite

Nitrate and some nitrite from food

Figure 4:The Entero-SalivaryCirculation cycle showingthe complex interconversionof nitrate, nitrite and NO inthe body and theconcentration andrecirculation of nitrates inthe salivary glands. (FromLundberg et al., 2008)

NOx Species

Nitrate

Nitrite

Nitric Oxide

Concentration inBlood (nmol/L)

Half Life NOx Species

20,000 - 50,000

100 - 500

Less than 1

5-8 Hours

1-5Minutes

1-2Miliseconds

From diet or fromthe oxidation ofnitrite in the body

From diet, nitrate,or from the oxidation of NO

From nitrite in thebody

Table 1: Comparing Nitrate, Nitrite and NO (from Hord et al.,2009). See Box 1 for an explanation of terms used in this table.


distant sites in the body?” The answer, it seems, lies inthe fact that NO is converted back into nitrite andnitrate. Although NO lasts only a short time and mayonly act directly and locally before it is oxidized intonitrite and then nitrate, this rapid oxidation to bothnitrite and especially nitrate, allows these much morestable molecules to then travel to distant sites for re-conversion back into NO as and when the specifictissue requires. In effect, both the nitrite and nitrate actas pro-drug forms of NO; in other words, nitrate andnitrite act as a stable reservoir and a depot for futureNO requirements by all cellular tissues throughout thebody. In this manner NO can act both locally and atdistant sites in a similar manner to various hormonessecreted by the thyroid or adrenal glands.

An interesting feature of nitrate in the blood is thatapproximately 25% of the nitrate is concentrated backin the salivary glands via the entero-salivary circulationcycle (See Figure 4). The concentration of nitrate in the

salivary glands is 100-fold greater than in the bloodand that of nitrite it is 1000-fold greater! The rest ofthe nitrate is eventually excreted in the urine. So bothurine and saliva are particularly rich in nitrate andnitrite. It is interesting then why animals lick theirwounds or that urinating on an injury is consideredbeneficial – the potential benefits of these behaviourswill be discussed in detail in later articles in thismagazine.

In summary the opposite biochemical processes ofoxidation and reduction of L-arginine and nitratebioconversion into NO respectively may be viewed as“ying-yang” with the more conventional L-Argininepathway occurring in conditions when there is plentifulof oxygen and the nitrate pathway as a “back-up”system occurring in conditions when there is lack ofoxygen. The key point is that without this vital back-upsystem our bodies would not be able to carry out anumber of biological functions.

REJUVENOXREDUCES STRESSAND BOOSTS ENERGY

TECHNOLOGY


AntioxidantAnti inflammatory

VASODILATOR

Lowers BP

PreventsUlcers &

Infections

Gastric MucosalProtection

Diuretic

DIET

NO

Nitrates/NitritesHigher or Low

Oxygen

Improves oxygendelivery to the body

Benefits conditionslike sickle cell disease

Helps arthritis,joint pain &

inflammation


Prevents ClotsPrevents

LDL Oxidation

Anti-platelet

Anti-bacterial

PreventsHeart

Atttacks

PreventsStrokes

PlateletsCholesterol

PreventsArterial Plaque

L-ArginineHigh Oxygen

Only

PreventsUlcers &

Infections

THE ROLE OF NITRATES IN THE BODY


NITRIC OXIDEAND THECARDIOVASCULARSYSTEM

Cardiovascular Disease (CVD) which includes highblood pressure (hypertension), angina (ischaemia),arterial damage (atherosclerosis), stroke and heartattack is still the number one cause of morbidity andmortality world-wide. One in three Americans isaffected by CVD, and over a billion people worldwideare affected by high blood pressure alone. In Canada,someone dies from heart attack or stroke every 7minutes, with CVD accounting for 30% of all deaths inCanada annually. The costs associated with CVD arequite staggering with 2006 cost estimated in the EECto be over 200 billion Euros. In Canada, the costsassociated with CVD in 2009 were estimated at $22.2billion in physician services, hospital costs, lost wagesand decreased productivity.

In recognition of these alarming statistics there is aserious initiative on the part of the government toincrease the daily intake of fruits, vegetables and fiberas well as to encourage people to exercise more. InNorth America, the previous recommendation of fiveservings of fruits and vegetables has been upgraded toa recommended nine servings per day.

Various epidemiological and cohort studies haverepeatedly and unequivocally reported a lowerincidence of CVD with higher intake of fruits andvegetables. These findings have culminated in thefamed Dietary Approaches to Stop Hypertension(DASH) study. The DASH diet was specifically designedto lower blood pressure and recommends eight to tenservings of fruits and vegetables a day, along withconsumption of low-fat dairy products. The results ofthe DASH diet in reducing blood pressure have been

found to be as effective as a single prescriptiontherapy for blood pressure!

Researchers from Johns Hopkins University reportedan 18% reduction in the 10 year risk of heart diseasewith the DASH diet. The researchers stated that, “Inaddition to reducing blood pressure, the DASH dietshould substantially reduce the risk of coronary heartdisease”.

Similarly, the lower incidence of CVD in patientsconsuming Mediterranean, vegetarian and Japanesediets has been attributed to the high fruit andvegetable intake associated with these diets. In factsome researchers have reported that the greatestprotection against CVD is offered by diets with thehighest content of leafy green vegetables (See Table 2).This has led scientists to ask some important questions;“What is the common denominator in such diets?” andtherefore “What are potential CVD protectivecompounds associated with these diets?”

It appears that the riddle has been finally solved. Agroup of researchers principally from Sweden, the UKand the US have independently and collaborativelyshown that the nitrate and nitrites in our fruits andvegetables may be the answer and have forwardedconvincing evidence to support their hypothesis.

Are Inorganic Nitrates the Answer?Nitrates can be classified into two categories: organicand inorganic. Organic nitrates are what most of usare familiar with; for example glyceryl trinitrate (GTN)is what is in the tiny tablets usually dispensed bypharmacists in glass vials and recommended to beplaced under the tongue for anginal pains. Anothercommon organic nitrate compound it isosorbidedinitrate, which is a more advanced and stable form ofGTN. These organic nitrates have been in use since themid 1800’s. Then there are the inorganic nitrates,

NITRIC OXIDEAND THECARDIOVASCULARSYSTEM


which include potassium nitrate and sodium nitrate.These inorganic nitrates are commonly found as saltforms in soils, rocks and plants and have been usedfor thousands of years in gun powder, foodpreservation and for heart ailments by early Chinesephysicians.

A recently discovered 1000 year old Chinesemanuscript translated by scholars at CambridgeUniversity shows the use of inorganic nitrates byBuddhist monks for heart ailments! (See Figure 5) It isquite remarkable that the Chinese physicians knew notonly about the inorganic nitrates but more importantlythey understood their proper use as evidenced by theiradvice to their patients to “Administer under tongueand have patient swallow the saliva” It is as if thephysicians knew back then about the entero-salivarycirculation of nitrate!

Figure 5. A medical recipe from Buddhist monks at Dunhuangfrom medieval times showing that the benefits of nitrates wererecognized as early as 900 years ago. (From Lundberg et al.,2008)

Dr. Nathan Bryan and his colleagues have investigatedthe nitrate/nitrite content of various herbs andvegetables consumed in the West and by the Easterncultures. Interestingly, they have found that herbs andplants that have been associated with a reduction inCVD are also those with the highest nitrate/nitritecontent. For example the herb Radix miltiorrhizae, alsoknown as Dan Shen, has a very high nitrate contentand is a widely used Traditional Chinese Medicine(TCM) for conditions like angina, myocardial infarctionor stroke. Its therapeutic efficacy has been associatedwith its ability to dramatically raise NO levels. Clinicalstudies have shown that Dan Shen’s therapeuticbenefit is nearly comparable to GTN. Furthermore,Dan Shen does not produce the tolerance thatnormally develops with most organic nitrates;inorganic nitrates seem to be free of this restriction.

Similarly, Bryan’s research group has looked at variouscommonly consumed western vegetables and their

nitrate/nitrite content. As can been seen from Table 2the vegetables with the highest nitrate/nitrite contentare spinach, cabbage and Bok Choy. Othervegetables such as beet root, collard greens and leeksalso have very high nitrate content. Not only are thesevegetables loaded with nitrate but many of them alsohave a high content of the enzyme nitrate reductace

which assists the conversion of nitrate into nitrite,thereby facilitating the conversion of nitrite into NO.This is particularly true for the Eastern herbCinnamomum. This herb is rarely used by itself for

CVD because it has a fairly low nitrate/nitrite content.However, when it is combined with herbs like DanShen, which do have high nitrate content, the effect ispronounced! In this combination, Dan Shen providesthe high nitrate content and Cinnamomum providesthe necessary enzyme to reduce nitrate into nitrite andeventually to NO for therapeutic efficacy.

Table 2: The nitrate/nitrite content of various widley consumedwestern vegetables


Putting under the tongue to causeheart ”qi” to flow freely.

For treating symptoms such as struck by evil,acute heart pains and cold in the hands and feet

which can kill a patient in an instant.Look at the patients fingers and those with

greenish-black nails are such cases.Take potassium nitrite (5 measures of a bi spoon)

and arsenic sulphide (1 measure of a bi spoon)and combine the two into a fine powder.

Lift the patients tongue and sprinkle 1 measureof a bi spoon under the tongue.

If saliva is produced have the patient swallow it.This is a sure cure.

VEGETABLENITRITE

CONTENT(mg/100g)

NITRATECONTENT(mg/100g)

Root Vegetables

Green Vegetables

Cabbage

Nightshade

Melon

Carrot

Mustard Leaf

Lettuce

Spinach

Chinese Cabbage

Bok Choy

Cabbage

Cole

Wax Gourd

Cucumber

Eggplant

0.002 - 0.023

0.012 - 0.064

0.008 - 0.215

0 - 0.073

0 - 0.065

92 - 195

90 - 95

12.3 - 267.8

23.9 - 387.2

42.9 - 161

0.009 - 0.242

0 - 0.041

0.364 - 0.535

0.001 - 0.006

0 - 0.011

0.007 - 0.049

102.3 - 309.8

25.9 - 125.0

76.6 - 136.5

35.8 - 68.0

1.2 - 14.3

25 - 42.4


NO and Reduction of Blood PressureHypertension or high blood pressure is defined as aSystolic Blood Pressure (SBP) of 140 mmHg or higheror a Diastolic Blood Pressure (DBP) of 90 mmHg orhigher, with normal blood pressure being defined as120/80 mmHg or lower. However, the risk of negativehealth effects and death is increased in individualswhose blood pressure is consistently 115/75 mmHg orhigher, with the risk of cardiovascular disease doublingwith each increment of 20/10 mmHg above 115/75mmHg.

High blood pressure is associated with an increasedrisk of stroke, myocardial infarction (heart attack),heart failure, kidney failure and cognitive impairment.SBP above 115 is the most important determinant ofthe risk of death world-wide and is responsible for 7-8 million deaths annually and over 20,000 deathseach year in the United States. In Canada more thanone in five people (22.7%) of Canadian adults aged20 years and older were living with diagnosedhypertension. Since approximately 17% of individualswith hypertension are not aware of their condition, thetrue prevalence of hypertension is likely higher. Thestatistics in the US are similar, with approximately 22%of adults living with diagnosed hypertension. It hasbeen suggested that in moderately hypertensivepatients a 5 mmHg reduction in blood pressure mightreduce the incidence of stroke by 22 % and coronaryheart disease by 16 %!

The beneficial effects of fruits and vegetables on bloodpressure are well known. For example, a comparisonof the effect of a traditional Japanese diet (with a highnitrate content) to that of a diet with low nitrate content(as much as 20-times less nitrate) in the same 25subjects (a cross-over study), demonstrated asignificantly lower DBP by 5 mmHg.

Because vegetables are naturally rich in nitrates itseems reasonable to investigate whether theconsumption of inorganic nitrate alone, correspondingto the amount present in a plate of salad for example,could affect blood pressure in healthy subjects. Dr. JonLundberg and Dr. Eddie Weitzberg’s group in Swedentested exactly this hypothesis in a small double-blindplacebo controlled, cross-over designed study. Sodiumnitrate in a dose of 7 millimoles of nitrate (see page 2:What is a mole?) per 70 kg adult male wasadministered for three days after which blood pressurewas measured. It was found that DBP was indeedreduced by a significant 4 mmHg compared to theplacebo (sodium chloride with equivalent amount ofsodium). The researchers found that the rise in

plasma nitrate levels resulting from consumption ofsodium nitrate was accompanied by a reduction inblood pressure. This suggests that the blood pressurelowering effect was due to the nitrate being convertedinto nitrite and then into NO. Although such changesin blood pressure appear relatively small, it must beremembered that the dose of nitrate used was notlarge; equivalent to a single serving of salad(approximately a plate or 250 g). Even so, these smallresults could still have meaningful health effects whenconsidered over the lifetime of the individuals. Forinstance, over the long term, even a small reduction inblood pressure will help reduce the stress put on bloodvessel walls due to high blood pressure. Additionally, itis important to consider that the study was conductedin healthy patients with normal blood pressure levels.It is expected that a significantly greater fall in bloodpressure would be observed in hypertensive patients.

Following up on the important findings of the Swedishresearchers, Dr. Amrita Ahluwalia and her group inLondon conducted an elegant study to prove the pointthat the nitrate rich vegetables can reduce bloodpressure via conversion into NO. Again in a double-blind randomized placebo controlled study, healthyadult volunteers were given 500 mL of beetroot juiceto drink. The amount of nitrate present in the juice wasthree times the amount administered in the Lundbergstudy. Three hours after consuming the beetroot juicethere were significant reductions in both SBP and DBPof 10 mmHg and 8 mmHg respectively. The beauty ofthis study was the clear demonstration of a cause andeffect relationship between the nitrate/nitrite and thereduction in blood pressure. By monitoring plasmanitrate and nitrite levels as well as blood pressurelevels, the researchers were able to show thatincreased plasma nitrate and nitrite levelscorresponded directly to a decrease in blood pressure.

NitriteNitrateSystolic bloodpressure

TIME (H)

3

400 0.8

0.4

0

200

0

0

-5

-10

6 12 24

Bloo

d ni

trite

conc

entr

atio

n (μ

M)

Bloo

d ni

trat

eco

ncen

trat

ion

(μM

) Ch

ange

in s

ysto

licbl

ood

pres

sure

(mm

Hg)

Figure 6. Association of changes inplasma nitrate and nitrite concentrationwith blood pressure. (from Kapil et al.,2010)


There are three noteworthy points that are raised bythis study. First, the half to one hour delay that occursbetween the intake of juice and the reduction in bloodpressure represents the time taken for nitrite to beconverted to nitrate by the bacteria in the mouth andthen to be converted into NO in the stomach. Second,the study demonstrates a dose response relationship,meaning that the higher the dose the greater theeffect. Finally, an exquisite relationship was establishedbetween raised plasma levels of nitrate/nitrite andblood pressure reduction. For scientists there is nobetter evidence than a physiological cause-and-effectrelationship, and this study accomplishes this!

A second study by Dr. Ahluwalia’s group providedfurther evidence, and also demonstrated that theblood pressure effect they observed could be achievedwith a lower 250mL intake of beetroot juice. Moreimportantly, they showed that the effects of dietarynitrate from beetroot juice can be sustained over alonger period, with the reduced blood pressure effectslasting for over 24 hours!

Lundberg and Weitzberg’s group have also usedanimal hypertensive models to provide furtherevidence of the benefits of inorganic nitrates forreducing blood pressure. In one of these models they

blocked the NOS enzyme (responsible for NOproduction via the L-Arginine pathway) with specificenzyme inhibitors of the enzyme. As a result ofblocking this enzyme the animals developedhypertension. This effect was reduced by administeringnitrites in the drinking water. In a more recent 2011study, this research group has shown thatadministering nitrates to hypertensive animals (causedby removing the kidney) not only reduced bloodpressure but also reduced scarring and other damageto the tissues in these animals. Furthermore, a muchlower dose of nitrite that did not reduce blood pressurestill protected against kidney damage, suggesting theuse of much lower doses of nitrates as a preventivemeasure. More studies are needed in this excitingfield.

Other CVD Protective Effects of Nitrates,Nitrites and NOIn addition to their blood pressure lowing effects,inorganic nitrates have been shown to have numerousother cardiovascular protective effects includingimproved endothelial function, improved plateletfunction, a reduction in the symptoms of metabolicsyndrome and improved oxygen delivery to the tissues.

A Note on Sunlight: Is it good for your Heart?In a recent publication of the European Heart Journal Dr. Martin Feelisch takes a deeper look at this question. Although thereis a definite relationship between UV light as a predisposing factor for non-melanoma and melanoma skin cancers especiallyin light skinned people, there is fair amount of data suggesting that exposure to sunlight can reduce blood pressure as wellas offering other health benefits. Previous researchers have attributed these benefits to the formation of vitamin D andmelatonin by the skin, however, Dr. Feelisch presents a novel hypothesis to explain this phenomenon. He suggests that thereis also a benefit due to the skin acting as a significant storage site for NO in the form of nitrate and nitrate from the diet. Infact the skin has a significantly higher nitrite concentration than the blood.

A recent human study has demonstrated that UVA radiation can increase plasma nitrite levels by over 40%. A similarincrease in animals has been shown to offer significant cardiac and circulatory protection. In addition, all-cause mortality riskis tightly related to latitude, and seasonal variations in light intensity are correlated with seasonal variations in the incidenceof CVD-related events and deaths. Rates of strokes, angina and heart disease are all higher in the winter months thansummer. Of course other factors like diet and exercise need also to be taken into account. Dr. Feelisch argues that since nitrateand nitrite levels are under dietary control it could be possible to greatly enhance plasma levels and thereby allow the bodyto produce NO on demand, where and when the body desires to achieve optimal health benefits.


The human circulatory system is the largest organ inthe body. The health of this enormously importantsystem depends on the proper functioning of a specifictype of cell that lines the inside of the blood vessels.These cells are called endothelial cells, and they formthe lining of every blood vessel in your body, from thelarge aorta to the tiniest capillaries. The endotheliallayer is only one cell thick and plays a vital role in thehealth of the blood vessel. Any abnormality ordamage leads to these cells leads to a condition calledendothelial dysfunction, which is thought to be a keyinitiating factor in the pathology of CVD.

High blood pressure, inhaled or ingested toxins (e.g.tobacco smoke), excessive alcohol intake, poor diet(e.g. excessive cholesterol or sugar) and stress can all

cause damage to the fragile endothelial layer. Forexample, high blood pressure results in an excess ofmechanical sheer forces to be exerted on the layer,which can damage the endothelial cells. On the otherhand, tobacco smoke may damage the cells bycausing an excessive generation of free radicals orreactive oxygen species. As the name implies these arehighly reactive molecules, and they can attack theendothelial cells, damaging them. Similarly, poor dietin the form of excessive consumption of sugar orcholesterol can initiate endothelial damage. In eachcase, the net result is the formation of plaques in theblood vessels. These plaques are highly dangerous asthey can cause the rupture and release of blood clots,resulting in further damage to distant sites and can

HealthyVessel

DiseasedVessel

Endothelial DysfunctionBuild up of Arterial PlaqueReduced Vessel ElasticityDecreased Blood FlowDecreased Oxygen DeliveryDecreased Limb Function

Nitrite &NO

Improved Endothelial FuntionIncreased Vessel ElasticityIncreased Vessel GrowthIncreased Blood FlowIncreased Oxygen DeliveryIncreased Limb Function

Figure 7. A comparison of a normal healthy blood vessel and a diseased showing damaged endothelial cells and abuild-up of atherosclerotic plaque. The figure shows the beneficial effect of nitrite therapy on diseased blood vessels.(from Patillo et al., 2011).


also lead to blood vessel blockage - causing heartattacks or strokes. Once the damage to endothelialcells begins; it is like a chain reaction, with thedamage spreading from endothelial cell to endothelialcell. Essentially, one plaque breeds more plaques,causing inflammation to spread like wildfire andcontributing to atherosclerosis of the blood vessel.Atherosclerosis is a build-up of plaque inside thearteries, which can thicken and harden over time (seeFigure 7, next pg). This results in a loss of blood vesselelasticity and flexibility which is critical to how theblood vessel responds to pressure changes. Any bloodvessel that has reduced elasticity will have increasedpressure inside it.

Endothelial cells are also important because they areone of the key sites in the body where NO issynthesized. NO acts as a local signaling moleculeand hormone to the neighboring cells, allowing formonitoring of the environment in the vessel andensuring that proper blood pressure and other factorsare maintained. NO also has direct actions on theblood vessel itself. It can travel to the smooth musclecells located directly beneath the endothelial cell liningwhere it caused vasodilatation or widening of theblood vessels. In a situation where excessive freeradicals are present, NO is unable to do its jobproperly since the free radicals will act to “mop” up theNO, reducing the amount of NO that is able to act tocause vasodilatation, which is not a good thing!

Studies using genetic knockout mice (see page 2) thatlack the endothelial NOS enzyme have shown thatthese animals are highly prone to cardiovascularcomplications. However, nitrite supplementation wasable to reduce such endothelial disturbances and helpto prevent resulting damage. The Ahluwalia studydiscussed above showed a similar beneficial effect inhumans. They found that beetroot juice effectivelyreduced endothelial damage as assessed by variousinflammatory markers like C-Reactive Proteins (CRP).Recently, a direct relationship between reducedendothelial damage and plasma nitrite levels hasbeen established in humans (see Figure 8). Overall,the data points towards a very important role fornitrate/nitrite in the preservation and support ofhealthy endothelial function.

Platelet functionPlatelets are cells present in the blood that are distinctfrom the other two major blood cell types, red andwhite blood cells. The primary function of the plateletsis to aggregate at sites of damage causing clotting.Platelets are able to cause this clotting due to their

innate tendency towards “togetherness’, in otherwords they tend to aggregate or clump together. Innormal circumstances this is a good thing, since theclotting caused by platelets is required to preventexcessive blood loss when an injury occurs. However,platelet adhesion, activation and aggregation are alsoknown to be key factors in the progression ofatherosclerosis and thus, modulation of plateletfunction is an important therapeutic strategy for theprevention of atherosclerosis. Both beetroot juice andpotassium nitrate have been shown to reduce plateletaggregation. Reduced platelet aggregation or“stickiness” not only helps to prevent the inappropriateclumping of cells that can lead to colt formation andartery blockage, but also allows blood flow to takeplace more easily thereby reducing the burden on theheart.

Figure 8. The association between blood nitrite levels andendothelial dysfunction. Healthy individuals show high nitrite levelswhich are associated with high levels of artery elasticity. Individualswith endothelial dysfunction show low nitrite levels, reduced arteryelasticity and thicker artery walls.

Metabolic SyndromeIn 1985, Reaven coined the term Metabolic Syndrometo describe a cluster of symptoms that often appeartogether and may have a common initiating orcausative event. This cluster of symptoms include:obesity, hypertension, high lipid/cholesterol levels anddiabetes. These deadly conditions are the fourhorsemen of the apocalypse that have targeted onequarter of the world’s adult population.Collectively, these four conditions act to increase therisk of CVD in a synergistic fashion, meaning that therisk increases exponentially rather than additively asmore and more factors become involved.

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Animal studies using knockout mice lacking the NOSenzyme gene (thus preventing NO formation) result infull-blown metabolic syndrome. Various researchershave suggested that metabolic syndrome may be dueto a deficiency of NO. Addition of nitrate and/or nitriteto the drinking water of these knock-out mice has beenshown to protect the animals, significantly reducestheir lipid and triglyceride levels and also reduces theincidence of diabetes and weight gain. These resultspresent an intriguing possibility for the prevention andcontrol of metabolic syndrome in humans, althoughhuman studies are required to verify these results.IschemiaIschemia occurs when there is a lack of oxygendelivery to tissues, often due to partial or completeobstruction of blood flow. For example, endothelialdysfunction leading to atherosclerosis can result inischemia. Angina (chest pains), heart attacks andstrokes are examples of ischemic events. Whenischemia occurs, the tissues that are deprived of bloodand oxygen suffer irreversible damage which canresult in death. Tissues like the heart, the kidneys andthe brain are particularly sensitive to an interruption inblood supply. Besides helping to reduce the incidenceof ischemic events in the first place through its abilityto widen the blood vessels (thereby improving oxygendelivery to the tissues) and through its improvement ofendothelial function, NO can also have direct effectsin the prevention of ischemic injury. Animal studieshave shown that during ischemic events, low oxygenacidic conditions result, which causes nitrite to be

reduced to NO. This production of NO duringischemic events is the body’s way of protecting thecells and tissues during periods of reduced oxygensupply. Studies in mice and rats have shown that aninfusion of nitrite during an ischemic event cansignificantly improve recovery and reduce tissuedamage and death in the heart, lungs, kidney andliver. Other animal studies have also found thatpretreatment with inorganic nitrate helps to enhanceblood flow to damaged areas following an ischemicevent, which can help to minimize damage. Overall,the evidence points to a preventative and protectiveeffect of nitrates and NO for ischemia. Further studiesin humans will provide further support for the role ofnitrates in these conditions.

Metabolic Syndrome:Are you at risk?

Metabolic Syndrome is characterized by the followingcollection of symptoms:

For Men:• Waist circumference > 40 inches • Triglycerides > 150 mg/dL

• HDL Cholesterol < 40 mg/dL • Blood Pressure > 130/85 mmHg• Fasting Glucose > 100 mg/dL

For Women: • Waist circumference > 35 inches • Triglycerides > 150 mg/dL

• HDL Cholesterol < 50 mg/dL • Blood Pressure > 130/85 mmHg• Fasting Glucose > 100 mg/dL

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NNIITTRRIICC OOXXIIDDEE,,EEXXEERRCCIISSEE AANNDDEENNDDUURRAANNCCEEIt is widely recognized that exercise improves aplethora of cardiovascular symptoms includingcholesterol, blood pressure, risk of stroke, vascularinflammation, and many others. Furthermore,researchers in the field of Applied Physiology andSports Medicine have found that exercise alsoimproves the function of the heart, accelerates cardiacrehabilitation, controls congestive heart failure (acondition where the heart is unable to pump the bloodefficiently) and prevents intermittent leg pains causedby poor blood flow due to blockage and hardening ofthe arteries.

Recent research seems to suggest strongly that thebenefits of exercise on cardiovascular health are inlarge part due to raised NO levels. Exercise increasesthe production of NO by the endothelial cells. SinceNO is a potent vasodilator and increased productionof this molecule causes blood vessels to becomerelaxed and widened, thereby allowing more bloodflow and increased oxygen delivery to the body’stissues. Lack of exercise, improper diet and lifestyleand ageing will all eventually lead to reduced NOgeneration at the tissue level, and particularly in theblood vessels, resulting in a consequent worsening ofcardiovascular symptoms.

Although it is acknowledged that exercise positivelyinfluences NO levels one wonders if the reverse is alsotrue. Namely can higher NO levels improve exercisecapacity?

Again researchers at University of Exeter in the UK andthe Karolinska Institute in Sweden have looked into thisphenomenon in great detail and have independentlycome up with some surprising findings! Essentiallythese researchers have found that raising NO levelscan indeed have a positive impact on exercise capacityand endurance!

For example, Lundberg and Weitzberg’s group inSweden conducted a small human randomizeddouble-blind-placebo-controlled study, where healthymale athletes were given a dose of 7 millimoles ofinorganic nitrate for three days prior to an episode oflow-intensity exercise on a cycle. This dose of thenitrate corresponded to the amount of nitrate thatwould be found in a plate of salad.

The researchers showed that supplementing with eventhis low dose of nitrate significantly improved theperformance of the athletes as measured by amountof oxygen being consumed (Vox). In other words,consumption of nitrates reduced the body’s demandfor oxygen during exercise. Furthermore, in addition toreducing the body’s oxygen requirement, or “oxygencost”, the researchers also noticed that the time takento reach exhaustion was also increased. This suggeststhat consuming nitrates prior to exercise can actuallyreduce and/or prevent exercise fatigue! In a follow-upstudy by the same research group showed that nitratesacted in the same positive way when athletesperformed high intensity exercise as well!

These findings were further supported by the Britishresearchers at Exeter University. Led by Dr. NigelBenjamin, one of the pioneers of the nitrate/nitrite/NOresearch, this group used beetroot juice instead ofsodium nitrate. The effect of beetroot juice was evidentas quickly as 1 hour after consumption, and reachedmaximum effect after three hours. The studies usingbeetroot juice confirmed the findings that nitratesreduce oxygen consumption and fatigue duringexercise. A second study by the same British groupshowed a similar effect when beetroot juice was takenfor a longer time period of 15 days. In other words,the positive effects were still evident when nitrates werebeing consumed on a longer term basis. This was notsolely an acute phenomenon!


Interestingly, both the British and Swedish studiesnoted that there was no increase in production of lacticacid in the athletes consuming nitrates! Lactic acidproduction normally occurs following exercise and isone reason why athletes become fatigued. As oxygenbecomes rate limiting, and tissues start becomingstarved of oxygen, the body resorts to an alternative,less efficient mechanism of energy production thatproduces lactic acid as a by-product. However, thesestudies demonstrated that in the presence of NO,energy production in the muscles remains efficient,which in turn improves the body’s ability to toleratehigh intensity exercise for longer time periods. It hasbeen shown that nitrate supplementation prevents thedepletion of a key muscle protein calledphosphocreatine; phosphocreatine measurements arean indicator of energy usage in the form of ATP.Research results have suggested that nitrates causemuscle contractions to use less energy.

Figure 9. Graph showing improvements to mitochondrial functionassociated with an increased intake of dietary nitrate. Highernitrate levels were shown to improve two key measures ofmitochondrial function: the efficiency of energy (ATP) production(as measured by oxidative phosphorylation ratio) and also themaximal energy (ATP) production. This means that increasingdietary nitrates enables mitochondria to produce more energymore efficiently!

Nitrite has also been shown to interact directly with themitochondria; the cellular powerhouses or energygenerating factories of the cells. Mitochondria produceenergy in the form of ATP, which is the energy currencythat all cell processes require to function. Dr. SrutiShiva at the University of Pittsburg School of Medicinehas studied the complex actions and interactions ofNO at the mitochondrial level and proposes that theunique exercise enhancing effects of inorganic nitratesand vegetables rich in nitrates may well be due to theirspecific interactions with various enzymes involved inenergy production in the mitochondria. Theseinteractions not only explain the exercise enhancingand oxygen sparing effects of NO but also help to

explain its cell protective effects. At the cellular level,NO acts to reduce the generation of reactive oxygenspecies, which prevents cellular and DNA damage andalso helps to prevent apoptosis or cell death.

In terms of athletic performance, the potential benefitsof inorganic nitrates are enormous. For athletes, thissmall, but important molecule may open the door forimproved performance without the negative healthand legal implications associated with other sportenhancing drugs!

Figure 10. Diagram showing the stress imposed by exercise ontissues like the heart and how nitrate/nitrite can increase NOavailability and protect the heart tissue when oxygen is limited

However, the significance of all of these findingsextends far beyond the field of exercise physiology. Forinstance nitrates could be of great benefit toindividuals suffering from reduced motility or exercisecapacity. The ability of NO to reduce muscle energyuse and extend the time to fatigue could be highlybeneficial to ambulatory convalescing patients, seniorsand heart patients who have a reduced exercisecapacity and require assistance in recovery times fromtheir illness.


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Blood Vessel

Increased production ofNO from L-Arginine

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Low oxygen levels cancause cardiac ischaemia

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THE HEART ISPROTECTED

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body mobilizes NOfrom its stored nitrite


Nitric Oxide andGastrointestinalHealthThe explosion of NO research in recent years has beenaccompanied by some provocative, brilliant and trendsetting findings about this simple molecule. NO playsa critical role in the health of the gut by protectingagainst various bacterial invaders and by maintainingthe integrity of the stomach lining, called the gastricmucosa. The gastric mucosa is a layer of mucous thatlines the inside of the stomach, and it essential forprotecting the cells of the stomach, and the rest of thebody, from the acidic environment within.

The human gastrointestinal tract (GIT) is a longwinding tube consisting of various types of cells fromthe mucosal cells in the mouth to the epithelial cells ofthe intestines and everything in between. Properfunctioning of the GIT is crucial for the maintenance ofoptimal health. However, the GIT and its cells mustconstantly cope with a complex assortment of friendlyand pathogenic bacteria, yeasts, toxins, chemicals,enzymes and nutrients like fats, carbohydrates,proteins and fiber. Further adding to the enormouscomplexity of this task is the fact that all of this musttake place across a wide range of pH levels, fromacidic, to neutral to alkaline.

Gut Microflora and NOIn terms of cell number, bacterial cells in the GIToutnumber human cells by a staggering 9 times! Thesebacterial cells encompass many different species andhave a wide range of functions – some of them friendlyand some of them detrimental. For example, somebacteria in the gut are responsible for the synthesis ofcertain essential nutrients like B vitamins, vitamin Kand folic acid, others are important for facilitating theabsorption of nutrients like calcium, and still othersproduce harmful toxins. Real estate in the GIT is highlysought after, resulting in intense competition betweenall the different bacteria for nutrients and space. Thetwo key sites with the highest density of the bacterialcells are the mouth and the large intestines.

Maintaining healthy colonies of bacterial florathroughout the GIT is essential for good health.Unfortunately, this delicate balance is easily disturbedby poor diet, an unhealthy lifestyle, the use of certainprescription medications like antibiotics and manyother factors.

Figure 11. The bacteria or “microflora” of your gastrointestinaltract. A healthy balance of these microorganisms is essential formaintaining good health.

In the mouth, saliva plays a critical role in not onlylubricating the food to facilitate swallowing but also inthe production of key enzymes that begin the digestiveprocess. Humans produce approximately 1 liter ofsaliva per day. This salvia is produced in the mouthand eventually makes its way into the stomach.Nitrates obtained from the diet (for example from theconsumption of lettuce, beetroot, spinach, leeks etc.)are rapidly converted into nitrite by a specific type ofbacteria housed on the surface of the tongue. Spittingor not swallowing the saliva following consumption ofthe above nitrate rich foods results in a lower nitriteconcentrations in the blood; hence the age-old butsensible advice, “eat your greens but don’t spit”.Similarly, the use of mouthwash also results in lowerblood nitrite levels due to the destruction of thebacteria on the tongue. It is therefore advisable not touse mouthwash immediately after eating vegetables,so as not to negate the benefits of these nitrate richfoods. A better strategy would be to drink a cup ofgreen tea after consuming a plate of salad or spinach;this is a healthy alternative that will not destroy theseimportant nitrate reducing bacteria. It is also importantto note that the conversion of nitrates into nitrite isenhanced by the presence of certain naturalcompounds like vitamin C, and antioxidants like the

INTESTINAL MICROFLORAINTESTINAL MICROFLORA1000 Billion Microorganisms comprising over 500 Different Species

Stomach

SmallIntestine

Large Intestine(colon)

Lactobacilli

StreptococciLactobacilli

EnterobacteriaEnterococcus

FaecalisBacteroides

BifidobacteriaPeptococcus

PeptostreptococcusRuminococcus

ClostridiaLactobacilli

1 to 10 thousand

100 thousand to 1 million

10 thousand to 100 million

10 billion to 10 trillion


polyphenols present in green tea, pomegranate,grape seed and other many other foods.

The nitrite molecule is actually a negatively chargedentity called an anion. In the past, nitrite wasconsidered to be an inert molecule with no biologicalsignificance. However, recent research suggests thatthe nitrite anion acts not only as a precursor to nitricoxide, but also as a suitable storage and transportform for future conversion into NO. This allows NO tobe produced in the body where and when it is needed.In addition, and somewhat surprisingly, nitrite andpossibly even nitrate have physiological benefits intheir own right. For example, in the mouth the nitriteanion has a powerful antimicrobial action that cansignificantly inhibit certain bacteria like Streptococcusmutans that are well known to contribute to thedevelopment of dental cavities. In this way, the actionsof the nitrite anion are similar to those of green tea.

As previously mentioned, a rather unique mechanismexists that allows the nitrate we consume to beconcentrated in the saliva through the processofentero-salivary circulation (see Figure 4). In thisprocess, nitrate taken in from a nitrate rich meal isabsorbed from the stomach into the blood and is thencirculated back into the saliva where nitrate isconcentrated and stored. Typically, saliva will have anitrate concentration that is at least ten-fold higherthan the blood and a nitrite content that is onethousand-fold greater than the blood! This helps toexplain why animas (and humans) lick their wounds;entero-salivary circulation is a natural means ofensuring that nitrate rich saliva can be converted tonitrite and then into NO for healing! In effect thenitrate in saliva is acting as a pro-drug, meaning thatit allows the delivery of NO in an easily useable andtransportable form. This is especially important due tothe much shorter half-lives (see Box 1) of nitrite and NO.

NO and the StomachThe acidic environment of the stomach is deliberatelyinhospitable and is designed to kill off ingestedpathogenic organisms like E. coli, Helicobacter pylori,Pseudomonas aeruginosa, Salmonella enteric,Yersinia, Shigella and others. The extremely low pH ofthe stomach ensures a direct killing effect of most ofthese bacteria, helping to prevent harmful infections.However, some bacteria like H. pylori, are resistant tolow pH and are able to survive the acidic environment.H .pylori is widely acknowledged to be the causativefactor of stomach ulcers, which in turn can lead to thedevelopment of gastrointestinal cancer. This bacterial

species is particularly resistant to acidic conditions andis very difficult to eliminate once infection occurs. Theonly known therapy is treatment with a cocktail ofpowerful antibiotics for periods of 6-8 weeks or longer.Even then the re-infection rate is fairly high. Thedamaging effects of H. pylori stem from its rather nastyhabit of borrowing deep into the gastrointestinalmucosal lining, which over time causes significantdamage and ulceration.

The good news is that NO has been shown to helpreduce the damage caused by this nasty bacteria.When nitrite rich saliva is swallowed, the highconcentration of hydrogen ions in the stomach (thelower the pH the higher the hydrogen ionconcentration) react very quickly with the nitrite,reducing it and thus converting it into nitric oxide. TheNO then acts locally on the stomach lining and startsthe healing process by reducing inflammation in thestomach. This is highly significant since ulceration andultimately gastric cancer are both inflammatoryconditions accompanied by the generation of a largenumber of damaging free radicals. In addition to itsanti-inflammatory and healing effects, NO alsoincreases blood flow to the stomach through its abilityto widen blood vessels. By increasing blood flow, NOimproves circulation and enhances delivery ofnutrients and key immune cells to the site of damage,facilitating and speeding up the healing process.Indeed, gastric NO formation from nitrite and nitratemay act as a first-line defense against swallowedpathogens.

In addition to reducing inflammation of the gastrictissue, the stomach also requires a continuous supplyof mucus in order to protect the stomach lining againstdamage from the acidic stomach contents. NO playsan important role in the maintenance of mucosalintegrity by stimulating the production of a proteincalled mucin, which is a key component of mucous.

H.PYLORI


Gastric NO protects against NSAID damageMost pharmaceuticals like aspirin or non-steroidalanti-inflammatory drugs (NSAID’s) have potentiallyserious side effects including gastric irritation, whichcan lead to excess bleeding in the stomach and evento the formation of gastric ulcers. This is in part due tothe inhibition of protective prostaglandins, a largegroup of chemicals that protect the gastric mucosa. Anumber of animal experiments have shown that lowNO is associated with a high incidence of gastricirritation. For example, in animals where the NOSenzymes responsible for production of NO wereinhibited, there was an increased incidence of ulcersand other gastric side effects. However, when theanimals were fed nitrate and then exposed to NSAID’sthere was significant protective effect, which wasattributed to an increased production of specificgastrointestinal protective prostaglandins. Thesestudies suggest that taking your NSAID’s or otherpotentially stomach irritating drugs like aspirin, withfoods rich in nitrates is a sensible option. In fact,pharmaceutical companies are on the verge ofreleasing drug formulations that combine NSAID’sand NO donors in order to help reduce the negativeimpacts these drugs have on the gastrointestinal tract.

When NO Production is Impaired

The NO3-NO2-NO reduction process can becomeinefficient under various conditions, especially as aresult of factors affecting the efficiency and normalfunctioning of the gastrointestinal tract. For example,the normal cycle of nitrate and nitrite storage and NOproduction can be impaired in seniors, patients inintensive care, patients on long term antibiotics,patients consuming too many antacids or patients notproducing very much energy.

Figure 12. Various factors that can negatively affect the efficiencyof the NOx3,2,1 pathway of NO production (from Weitzberg et al.,2010)

Disturbances of the pathway may occur at variouslevels; for example, reduced saliva production canimpair nitrite production, reduced stomach acidity canimpair NO production and high levels of free radicalscan act to scavenge NO before its beneficial actionsare realized. It may be advisable for patients in theseconditions to consider an increased consumption ofnitrates to help boost NO production.

Reduced dietary supply

Reduced saliva productionAntibiotics

Reduced gastric acidityReduced swallowing

Increased NO scavengingby free radicals

BIOACTIVITY

NO

NO

NO

3

2

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AAnnttiimmiiccrroobbiiaallaanndd HHeeaalliinnggEEffffeeccttss ooff NNOOIn addition to its role in eliminating pathogens in thegut, NO plays an important role in the prevention andmanagement of infections in other areas of the bodyas well. For example, NO can have directantimicrobial effect against a wide range of pathogensincluding those that cause urinary tract infections, skininfections and those associated with cystic fibrosis. Inaddition to this, NO stimulates the immune system toaccelerate wound healing and acts to improve bloodflow to the damaged areas, improving the immunesystem’s ability to deal with infections. These benefitsof NO are described in more detail below.

NO and Bladder infectionsUrinary Tract Infections (UTI’s) are fairly common inwomen, especially in young women aged 20-45years. The causative agent of these infections iscommonly the bacteria E. coli, which is a fairlycommon transient of the urinary tract. One effectivemethod of treating this condition is by using D-Mannose, a simple yet highly effective five carbonsugar (unlike the more common table sugar or fruitsugars which have six or twelve carbon atoms). D-Mannose works via a simple yet highly effectivemechanism. Essentially the sugar binds to the bacteria(by saturating all the binding sites or “hooks” on thesurface of the bacteria) which in turn prevents thebacteria from latching on to the urinary tract wall. Thebound-up bacteria are thereby safely flushed out ofthe system in the urine.

Unfortunately, in some patients the D-Mannosetreatment does not work or requires repeatedadministration of the sugar. A potential solution forthese unlucky individuals is nitrates! Over sixty percentof the nitrate and nitrite we consume is eliminated inthe urine, where it provides a direct antimicrobial

effect for the urinary tract tissue. This antimicrobialeffect is greatly enhanced if the urine is furtheracidified by vitamin C intake, which can be obtainedfrom drinking lemon or cranberry juice. The excessacid in the urine helps to enhance the conversion ofnitrate and nitrite into NO. The results of test tubestudies have suggested that the antimicrobial action ofnitrite with vitamin C is as effective as the prescriptiondrug nitrofurantoin, which is commonly used in thetreatment of UTI’s.

NO and Cystic FibrosisIn cystic fibrosis patients there is a buildup of fluid inthe respiratory tract due to excess chloride ionexcretion which often requires physiotherapyassistance to aid in eliminating the excess fluid.Unfortunately, there is also an increased tendency ofthe respiratory passages to be inhabited by pathogenslike Pseudomonas aeruginosa, causing chronicinfections and even death among these patients. Thistype of bacteria is highly resistant to antibiotics and isvery difficult to eliminate. Nitrates may prove to be abeneficial treatment for patients infected with thesebacteria. In animal models cystic fibrosis treatmentwith acidified nitrite has been shown to completelyeliminate this resistant pathogen.

The Role of NO in Skin Health and Wound RepairDr. Adrian Barbul and Dr. Maria Witte of Eberhard-Karls-Universität in Tubingen, Germany haveinvestigated the role that NO plays in wound repair fora number of years. Treatment of acute and chronicwound healing failure is still a major unresolved issuein medicine. Approximately twenty five percent of thedelays in hospital discharge can be attributed to failurein wound healing. Over the years Barbul and Witte’sfindings have suggested that increasing NO levelscould be the key to the promotion of proper woundhealing. The key results of their studies include thefollowing:

1. L-Arginine stimulates wound healing by increasingNO levels.

2. Animals with an L-Arginine free-diet showedreduced wound healing. This was attributed to lowNO levels.

3. Blocking the NOS enzymes with a specific inhibitordelays wound healing.

4. Providing nitrates and/or nitrites accelerateswound healing.


5. Plasma levels of nitrate rise during wound healingas evidenced by higher urinary nitrate excretion.

Wound healing is a highly complex process. Followingan injury, wound healing is stimulated in a very specificsequence of events. In the first stages of woundhealing white blood cells called macrophages releaseNO as a means of inducing the inflammatory processwhich is essential for the initiation of healing. Shortlythereafter, NO levels fall rapidly, corresponding to thedelayed proliferative and regenerative stages ofwound healing as shown in Figure 13. Adequatesupplies of NO are therefore required in order for thebody to quickly and efficiently initiate the healing ofwounds.

Figure 13. Phases of wound healing and the generation of woundNO. (from Witte and Barbul, 2002)

RegenerationProliferation

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A Note on the Hygiene Hypothesis: Soil, Bacteria, Nitrite and the SkinDr. Martin Feelisch and Dr. David Whitlock from the famed Massachusetts Institute of Technology have recently proposed an interestinghypothesis regarding the production of nitrite and NO on the skin. They propose that humans evolved with a specific bacterial microflora thatcontributes to the regulation of NO physiology. Essentially the researchers argue that nature designed our bodies to coexist with a certain amountof bacteria called ammonia oxidizing bacteria (AOB). Unlike the bacteria present on our tongue, which converts nitrate into nitrite using areductive process, the AOB actually oxidizes (the opposite of reduction) the ammonia that is secreted in our sweat to form nitrite. This nitrite notonly converts into NO but also acts as signaling molecule in its own right

The skin is one of the largest organs and represents a major depot and storage site for the AOB. The hygiene hypothesis states that as weendeavor to keep everything around us clean, we are exposed to fewer bacteria, and as a result have a reduced ability to handle bacteria. Thisresults in a weakened immune system and a greater chance of infection when we eventually do encounter infectious organisms. The situation ismuch like how our muscles will atrophy if we don’t exercise regularly. Modern skin hygiene practices, including excessive bathing, washing, andthe use of shampoos and soaps, have led to a gradual loss of AOB from our skin. Over time we have experienced a reduced exposure to these“old friends”, which have accompanied human evolutionary history for millennia. It is thought that this reduced exposure has lead to a skewedresponse by our immune system which causes it to overreact. This tendency towards an exaggerated immune response, along with other lifestylerelated changes (e.g. urbanization from rural areas, increased consumption of processed foods and reduced intake of nitrate rich foods e.g. leafygreen vegetables) has led to an epidemic of immune related disorders and infections.

Like the mouth and large intestine, the skin is a large repository of diverse organisms. Unfortunately, unlike the attention that probiotics andgut health have received over the years, little interest has been paid to skin microflora. Whitlock and Feelisch present convincing evidence aboutthe need of these bacteria for optimal health. These bacteria include the genus Actinomyces, which are abundantly present in soils and the waterin ponds, rivers and lakes. Dr. Whitlock has even proposed that the replenishment of skin bacteria may be one reason that many animals rollin soils and dirt regularly!

The presence of AOB on the skin is hypothesized to increase the body’s nitrite pool through the conversion of ammonia into nitrite and NO. Thisnitrite and NO can then freely diffuse across the dermal barrier and be transported via the circulatory system to distant sites. Of course, largerpopulation studies are warranted to test this hypothesis. For instance it would be interesting to see the results of a comparative assessment ofurban and rural populations and their skin AOB content, plasma nitrite levels and the incidence of immune disorders. Whitlock and Feelischargue that such studies may unveil a condition they term “nitropenia” or a deficiency of nitrate and/or nitrite due to a lack of AOB on the skin.They propose that this may, in turn, be the cause of numerous diseases and they suggest a possible treatment via topical AOB therapy. This isan intriguing hypothesis that warrants further testing.



Nitrates and nitrite also powerfully stimulate theimmune system to accelerate wound healing and toimprove blood flow to the damaged area. Finally,nitrates also assist in the restitution of the skin byenhancing collagen synthesis and regeneration.

Dr. Nigel Benjamin is one of the foremost researchersin the field of nitrate/nitrite and NO physiology. Basedin the UK at the University of Exeter Medical School,Dr. Benjamin has actively pursued research in diverseareas of nitrate research, including gut health, exercisephysiology, blood pressure and the topical use ofnitrates for various skin conditions.

In his studies on NO, Dr. Benjamin noted thatgenetically manipulated knock-out mice lacking NOSenzymes for generating NO demonstrated anincreased susceptibility to diseases like tuberculosis.This evidence adds to the growing body of researchsupporting the anti-microbial benefit of NO, and alsoled to a series of other studies examining the effects ofNO on various micro-organisms. Using sodium nitrateand nitrite combined with acidifying agents likevitamin C or citric acid, Dr. Benjamin and hiscolleagues have shown the effectiveness of NOagainst a large number of bacteria, fungi and otherpathogens. The list of susceptible organisms is longand impressive, including but not limited to, E. coli,Candida, Leishmania, Staphylococcus aureus,Propionibacterium acnes (acne causing bacteria),Trichophyton (which is the cause of Tinea pedis orathletes foot) and Mycobacterium ulcerans (Buruli

ulcer). A large number of these bacteria cause skinconditions, and Dr. Benjamin’s work has focused onthe use of topical nitrates to eliminate and treat theseconditions and infections.

One study of topical nitrates which was particularlyinteresting examined the potential of nitrate treatmentof Buruli ulcers (caused by Mycobacterium ulcerans), aserious ulcerating skin disease that is common inmany tropical countries. This particularly nasty ulcerhas no therapeutic treatment except for surgery whichinvolves skin grafting. In a 37 patient double-blindplacebo controlled trial, sodium nitrite was shown tobe highly effective, affordable and represented amajor advance in the treatment of this serious skincondition.

Topical NO therapy offers many intriguing possibilitiesfor the treatment of various skin conditions. However,it is unlikely to be effective in psoriasis since thesepatients have been shown to have very high NO levels,about a hundred to a thousand fold higher thanhealthy patients.

NO and other Biological EffectsNO is a simple molecule with the ability to cross-talkwith various cellular receptors and thus offers wide-ranging health implications. In the past nitrate andnitrite have been used as an effective diuretic, anantidote for cyanide poisoning, lung disease and otherwide-ranging applications.

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Nitrates have received some bad press in the past, butthe truth is that more and more research is showingthat they are a safe and healthy part of your diet. Forinstance, some of the healthiest foods, includingspinach, kale and beetroot, contain very high levels ofnitrates. Recent population and toxicological studieshave also shown that nitrates are important nutrientswith significant health benefits and without negativeeffects.

Despite the use of nitrates for centuries, there are twomajor areas of concern which are often brought upregarding their safety. These are the possibility ofmethemoglobinemia and the potential to cause cancerthrough the formation of nitrosamines. These twoissues are addressed below.

Do Nitrates cause Methemoglobinemia? Methemoglobinemia is a condition that occurs whennitrite binds to the iron in the hemoglobin molecule inred blood cells. Hemoglobin is an essential proteinthat binds to oxygen and then transports the oxygen tocells all over the body. In the 1950’s, a few cases ofcyanosis or blue-baby syndrome caused by a reducedability of oxygen to bind with hemoglobin weredocumented in infants. These occurrences wereimmediately attributed with nitrites and diagnosed asmethemoglobinemia. The infants were found to live inan area that had high levels of nitrite in the well water,and therefore it was thought that this was the cause ofthe reduced oxygen binding capacity of the babies’hemoglobin. While it is possible that this was the case,other factors were not taken into account. For instance,the possible contamination of the drinking water withbacteria was not investigated.

As a consequence of this diagnosis, governmentsaround the world quickly implemented strictregulations regarding the nitrate and nitrite content ofdrinking water, and took very expensive measures(hundreds of millions of dollars per year) to removenitrate from the water supply. The current limitimposed in the US, Canada and the UK is 50 mg or 1millimole per litre.

However, careful evaluation of the data does notsupport such rigid limitations. For example,commercial beetroot juice contains close to 20-45millimoles per litre of nitrate and measurements ofplasma levels following consumption of such highdoses show that levels remain within the physiologicallimit of 1 micromole per litre. It seems that the body isable to rapidly regulate nitrate levels and excess intakedoes not equate to dramatically elevated plasmalevels.

Figure 14. The limit of nitrate allowed in drinking water in Canadaand the US is 1 millimole per litre, however, a healthy glass ofbeetroot juice can contain up to 45 millimoles per litre!

Secondly, the research of Dr. Mark Gladwin in the UShas shown that when very high doses of nitrite aregiven intravenously to patients to prevent damage tothe heart, plasma levels increased to 16 times normalphysiological levels, yet the percentage ofhemoglobinemia was less than 1 percent! It seems thatthe body has various homeostatic mechanisms tomaintain physiological levels of nitrite. While cautionshould be exercised in not administering nitratesand/or nitrites to infants and/or children, researchindicates that in adults methemoglobinemia may notbe as serious as originally thought, especially when thedoses consumed are similar or less than those thatwould be found in a plate of salad or a glass ofbeetroot juice.

Do Nitrates form Carcinogenic Nitrosamines?The second potential cause for concern is theformation of nitrosamines. Nitrosamines arechemicals that can form between secondary aminespresent in foods and nitrite. Nitrosamines have longbeen associated with cancer formation in the liver.However, there are several shortcomings to the theorythat dietary nitrates can form nitrosamines in humans.

Glass of Water: Limited toless than 1 millimole per

litre of nitrates

Beetroot Juice: Up to 45millimoles per litre of nitrates;

associated with numerous health benefits!

The Safety ofInorganic Nitrates


First, the extrapolation of animal data to humans isquestionable when it comes to nitrates. Humans,unlike rats, have evolved mechanisms of concentratingnitrate in the salivary glands much we concentrateiodide ions in the thyroid gland. Second, nitrate isactively reabsorbed in the kidneys, suggesting anevolutionary advantage of concentrating this ion.Why else would evolution design such a conservationmechanism if such an ion was toxic? Third, a verycomprehensive 2003 WHO (world healthorganization) and joint expert committee on foodadditives concluded that: “overall the epidemiologicalstudies showed no consistently increased risk forcancers with increasing consumption of nitrate”.Further epidemiological and animal studies havefailed to find any conclusive correlation. Fourth,certain populations, for instance Tibetans living at highaltitude, have a hundred-fold greater plasma levels ofnitrates than those living at sea level without anyconcomitant increase in cancer rate.

In conclusion nitrate has been used in food andnutrition for centuries without evidence of negativehealth effects. It is especially telling that some of thehealthiest foods (like green, leafy vegetables) containvery high levels of nitrates, and that the high nitratecontent of these foods has been linked to their healthbenefits. As mentioned previously, researchers havesuggested that nitrates are probably responsible forthe health benefits associated Mediterranean andJapanese diets. Studies showing beneficial effects ofthese important molecules are extensive, and providestrong support for their benefits to health (see Table 3).

The ability of nitrate to increase NO levels quickly andeasily through the NOx3,2,1 pathway offers a novelapproach for maintaining optimal health. Nitric oxideis an important molecule that plays a key role innumerous and diverse physiological mechanisms.Furthermore, nitrate is an important nutritionaltherapeutic approach in preventing numerousdiseases by playing a key role in NO homeostasis.

Organ System Nitrate Source Species Effect Reference

Cardiovascular, Kidney Sodium Nitrate Rat Increased post-ischemic blood flow Jansson et al., 2008Cardiovascular, Stomach Sodium Nitrate Decreased MAP, decreased DBP, increased NO formation in the stomach, increased blood flow to the stomach Petersson et al., 2009Stomach Potassium Nitrate Rat Decreased ulcer formation, increased NO formation in the stomach, increased blood flow to the stomach Miyoshi et al., 2003Stomach Sodium Nitrate Rat Decreased ulcer formation, increased gastric mucus production Jansson et al., 2007Cardiovascular Potassium Nitrate Human Decreased platelet aggregation Richardson et al., 2002Cardiovascular Sodium Nitrate Human Decreased DBP, decreased MAP Larsen et al., 2006Cardiovascular Beetroot Juice Human Decreased SBP, DBP and MAP, reduced platelet aggregation, decreased endothelial function Webb et al., 2008Cardiovascular Potassium Nitrate, Beetroot Juice Human Decreased SBP and DBP Kapil et al., 2010Cardiovascular Traditional Japanese Diet Human Decreased DBP Sobko et al., 2010Cardiovascular,Musculoskeletal Sodium Nitrate Human Decreased DBP and SBP, decreased oxygen consumption during moderate exercise Larsen et al., 2007Cardiovascular, Musculoskeletal Beetroot Juice Human Decreased SBP, decreased oxygen consumption during moderate exercise, increased time to exhaustion during exercise Bailey et al., 2009Cardiovascular, Musculoskeletal Beetroot Juice Human Decreased DBP and SBP, decreased oxygen consumption during low intensity exercise, decreased muscle phosphocreatine, increased muscle efficiency Bailey et al., 2010Cardiovascular, Musculoskeletal Beetroot Juice Human Decreased DBP, SBP and MAP, decreased oxygen consumption during exercise Vanhatalo et al., 2010Musculoskeletal Sodium Nitrate Human Decreased oxygen consumption during maximal exercise Larsen et al., 2010Sickle Cell Disease Sodium Nitrate Human Increased regional blood flow Mack et al., 2008

Table 3: Therapeutic Effects of Inorganic Nitrate or Natural Sources Containing Nitrate. MAP = Mean artial pressure, DBP = DiastolicBlood Pressure, SBP = Systolic Blood Pressure. (From Weitzberg et al. 2010)


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