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  1. 1. ADVANCES VOLUME 3 • ISSUE 10IN ORTHOMOLECULAR RESEARCH 321 NOx TechnologyThe Power of Nitric Oxide 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 research-driven botanical integrative orthomolecular innovative
  2. 2. ADVANCES VOLUME 3 • ISSUE 10 IN ORTHOMOLECULAR RESEARCH 1. The Power of NOx: Say Yes to No! 8. Nitric Oxide and Cardiovascular Health 16. Nitric Oxide, Exercise and Endurance 18. Nitric Oxide and Gastrointestinal Health 21. Antimicrobial and Healing Effects of NO 24. The Safety of Nitrates 1 16 21 8 18 24 ADVANCES Advances in Orthomolecular Research is published and distributed through integrative physicians, health care practitioners, and progressive health food retailers. The content of this magazine is provided for informational purposes IN ORTHOMOLECULAR RESEARCH only, and is not intended as medical advice for individuals, which can only be provided by a healthcare professional. Contents and design © 2010 AOR. Any reproduction in whole or part and in print or electronic Published in Canada by Research & Writing form without express permission is strictly forbidden. Permission to Traj Nibber, BSc. MSc. Ph.D reproduce selected material may be granted by contacting the publisher. Advanced Orthomolecular Director of Research Research Inc. Questions? Comments? Graphic Design/Art Production Contact us at: Publisher/Editor-in-Chief AOR Inc. Neil Bromley 3900-12 St NE Megan Tracey, MSc Kyle David Boudreau Calgary, Alberta Canada T2E 8H9 e-mail or www.aor.ca2 Digital version of this magazine and back issues are available online at ADVANCES in orthomolecular research Volume 3 Issue 8
  3. 3. NO is now known to be a signaling molecule; The Power of meaning that is acts as a messenger to facilitate communication between cells, the relaying of messages and other cellular chatter. The body relies on a wide variety of signaling molecules including Nitric Oxide: hormones, nerve impulses and chemicals like cytokines, hydrogen and carbon dioxide. These signaling molecules help the body perform vital functions including the mobilization of the body’s Say Yes to NO! immune defense systems against invading foreigners like viruses, bacteria, toxins and carcinogens, as well as the modulation of other body functions like temperature and blood pressure. Besides being a simple molecule consisting of two of What is Nitric Oxide? the most abundant and well know elements, NO is also a radical. This means that NO has an unpaired Nitric Oxide (NO) is a simple molecule consisting of electron, making it a very reactive molecule! NO’s one atom of nitrogen and one of oxygen, making it unpaired electron means that it has extensive reactivity even simpler than water itself! Research into the effects with virtually anything and everything from body cells of this small but important molecule began in the and tissues to other chemicals and molecules 1970’s when scientists started to examine why blood circulating in the system. This reactivity is essential in vessels relaxed when certain compounds were added. enabling NO to carry out its many biological functions. This led to the discovery of NO and it’s amazing How is NO generated? effects in the body, with NO being named the The conventional method of NO synthesis is from the molecule of the year in 1992! In 1998, twenty-some amino acid L-Arginine (see Figure 1). L-Arginine is years after this research first began, a Nobel Prize was awarded to these researchers for their breakthrough oxidized via a series of steps involving a family of discoveries regarding NO. Unfortunately, one of the enzymes called nitric oxide synthases (NOSs). There pioneering researchers in the field of NO-research, are essentially three common NOSs termed isozymes; Salvador Moncada, was overlooked by the Nobel Prize these are iNOS, eNOS and nNOS. Each of these committee, showing that there is indeed a political side enzymes plays a different role in the generation of NO to science! Over the last two decades NO research has in different tissues like the nerves, endothelium or continued to grow, and there has been an exponential blood vessels or on demand. In each case however, increase in the number of publications on this normal oxygen conditions (also called “normoxia”) as fascinating molecule. well as a neutral to high (alkaline) pH level are Figure 1: The production of Nitric Oxide ( NO) from L-Arginine by enzymes called NO synthases (NOSs) Nitric Oxide Synthases (NOS’s) L-Arginine Nitric OxideVolume 3 Issue 10 ADVANCES in orthomolecular research 1
  4. 4. KEY TERMS AND DEFINITIONS Mediterranean Diet Consumption of fruits and vegetables has been known for some time to protect against Cardiovascular Disease (CVD) and Cancer. In the mid 1960’s an 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, saturated fats 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 of factors, 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 other four 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 corresponded to a reduced rate of cardiovascular disease. The Mediterranean diet is now universally recognized as a healthy diet. DASH Diet (Dietary Attempts to Stop Hypertension) is Oxidationmodeled after the Mediterranean diet.In the DASH diet an intake of fruits and vegetables Oxidation is a term that means an increased number of oxygen atomsof upwards of 8-10 servings per day is recommended. (strictly speaking it refers to the loss of electrons) for example when NO undergoes oxidation the number of oxygen atoms increases: NO NO2 NO3. Hypoxia Reduction Hypoxia is a condition when oxygen availability is greatly reduced. For example, such a condition is likely to occur during ischemia, Reduction is a term that is the which is when there is an interruption of blood flow to the tissues opposite of oxidation and refers to the loss of oxygen atoms NOS (e.g. the heart during a heart attack). (again strictly speaking it is the gain of electrons) NOSs are Nitric Oxide Synthases, for example when NO undergoes which are a diverse range of enzymes reduction the number of oxygen that facilitate the formation of NO atoms decreases: NO3 NO2 NO. from various starting molecules (substrates) like L-Arginine. Free Radicals Free Radicals are molecules that are very reactive due to an unpaired electron. Free radicals are generally Genetic Knock-Out considered to have negative health implications but that isn’t the universal case. For example, Genetic knock-out mice/rats are widely used to test NO itself is a free radical! what happens when a certain gene is deleted. When a gene is deleted the mouse can no longer produce a certain protein or enzyme. For example, using rodents lacking a specific gene that codes for NOS enzymes, one can test the effect of a lack of NO and its health implications. Bioavailability Bioavailability is a term that refers to the amount of an active molecule that reaches the target site of action. Poor bioavailability is a huge problem for large number of natural and pharmaceutical compounds. There is active research into various ways of delivering more of the molecule at lower doses. Mole Mole is a measurement widely used in chemistry that equates how much of the active molecule is present. The mole and/or milli-mole Reactive Oxygen Species (ROS) (mM - a thousandth of a mole) is widely used to express the concentration Reactive oxygen species (ROS) is another term of an active compound in the blood. for free radicals; species that are very reactive. Half Life Half-life is the time taken for the concentration of a compound to be reduced by half. Various metabolic processes in the body act 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. 2 ADVANCES in orthomolecular research Volume 3 Issue 10
  5. 5. required. When these conditions are met the NOS- reduced (see key terms pg 2) in the mouth by bacteria dependent conversion of L-Arginine occurs efficiently. that are normally present on the back of the tongue. Production of NO is the primary reason for the dietary These specialized bacteria use the nitrate to help them intake of L-Arginine. However, under low oxygen make energy in the form of ATP In return, the bacteria . conditions (also called “hypoxia”) the conversion of L- utilize their own nitrate reducing enzyme called Nitrate Arginine to NO is severely limited. Low oxygen Reductase to generate nitrite, which as we shall see is conditions can occur for a variety of reasons. For very useful to us! This special relationship is an interesting example of human-bacteria symbiosis: a example, partial or complete blockage of a blood mutually beneficial relationship. vessel (ischemia), conditions of extreme physical exercise or high altitude situations like mountaineering The nitrite is a much more active molecule than nitrate can all result in reduced blood flow and thus reduced and is present in high concentrations in the saliva which is swallowed into the stomach where conditions oxygen delivery to the body’s tissues and cells. of low oxygen (relative to the mouth) and high acid are Moreover, such low oxygen conditions are also present. These conditions are ideal for further accompanied by an increased production of lactic acid reduction of nitrite into NO. The entire reduction which reduces pH making the tissue condition both process of nitrate into nitrite and then into NO occurs hypoxic and acidic. As such, a body builder pumping without the intervention of NOS enzymes. These iron or an energetic aerobic class or a middle to long enzymes wouldn’t be active in these low oxygen and distance runner derive little if any benefit from L- low pH conditions anyway. It should be noted that low Arginine in terms of NO generation. oxygen and low pH conditions don’t just occur in the stomach, they can also occur throughout the body in certain situations including extreme physical exercise, A New Discovery! A Second Pathway to NO heart disease and psychological and physical stress! Generation: The NOx 3,2,1 pathway Both pathways of NO generation are depicted in However, there is good news for these athletes! Figure 3, below. Recently a novel pathway to NO generation from dietary nitrates has been discovered by researchers at L-Arginine Nitric Oxide the Karolinska Institute in Stockholm, Sweden and by (NO) Normal Low Oxygen Conditions Oxygen Conditions researchers from the University of London, England. (Normoxia) (Hypoxia) The Swedish researchers, led by Jon Lundberg and NOS- NOS- Eddie Weitzberg, along with Nigel Benjamin from Dependant Pathway Independent Pathway London, were trying to discover why certain diets, like Oxidation Reduction the Mediterranean diet, vegetarian diets, Japanese diets and the famed DASH diet (Dietary Advice for Stopping Hypertension) were particularly protective of Nitric Oxide Nitrate & Nitrite - - (NO) (NO 3 /NO 2 ) the heart. Both groups independently reported that the key to the success of these diets was the consumption of leafy green vegetables, and that a key component Figure 3. There are two ways that the body generates NO depicted as Ying Yang. Each pathway has special requirements. NO can be produced from of all these diets was the high nitrate content. The L-Arginine and oxygen by a family of enzymes, the NO synthases (NOSs). In a more recently discovered pathway, inorganic nitrate and nitrite are researchers proposed that the nitrate was converted reduced to form NO in blood and tissues when oxygen levels are low. into NO via a reductive process as follows: Although NO generation by the L-Arginine pathway is ineffective as oxygen levels fall, the nitrate–nitrite–NO pathway is enhanced! (From Lundberg et Essentially, the highly nitrate-rich vegetables are al., 2008) Figure 2: The conversion of dietary nitrates to nitrite and finally to nitric oxide. Conversion of nitrate to Reduction of nitrite to nitrite by bacterial enzymes NO in the stomach in the mouth or blood Nitrate (NO3- ) Nitrite (NO2-) Nitric Oxide (NO)Volume 3 Issue 10 ADVANCES in orthomolecular research 3
  6. 6. Oxidation is a step-wise increase in the number of See Table 1 for a detailed comparison between nitrate, oxygen and reduction is the opposite with oxygen nitrite and NO. being reduced from 3 to 2 to 1; hence the term All of this begs the question: “How is it that a molecule NOx3,2,1 pathway. Besides specific conditions like with such a short half-life can exert biological effects at oxygen status and pH, other factors like the presence of certain reducing compounds will also help to Table 1: Comparing Nitrate, Nitrite and NO (from Hord et al., enhance the conversion of nitrates into NO. These 2009). See Box 1 for an explanation of terms used in this table. helpful reducing compounds include vitamin C, alpha Concentration in lipoic acid and many polyphenols like green tea NOx Species Half Life NOx Species Blood (nmol/L) catechins (EGCG) and resveratrol. 20,000 - From diet or from Once NO is generated in the stomach it is rapidly Nitrate 5-8 Hours the oxidation of 50,000 nitrite in the body absorbed across the cell membrane into the blood vessels and from there it is carried all over the body. 1-5 From diet, nitrate, Nitrite 100 - 500 or from the However, as mentioned before, NO is a free radical Minutes oxidation of NO and is therefore a highly reactive species that lasts only a few milliseconds (a few thousandths of a second) Nitric Oxide Less than 1 1-2 From nitrite in the Miliseconds body before it is oxidized back into both nitrite and nitrates. Figure 4: Nitrate in the blood can also be The Entero-Salivary picked up by the salivary glands and start the cycle of NO Circulation cycle showing production all over again the complex interconversion of nitrate, nitrite and NO in the body and the concentration and Nitrate and some nitrite from food recirculation of nitrates in the salivary glands. (From Lundberg et al., 2008) Some nitrate and nitrite reach the blood and can be converted into NO when oxygen levels are low Bacteria in the oral cavity convert nitrate to nitrite The acidic environment of the stomach results in the conversion of nitrite to NO. This NO can go on to have effects throughout the body Any left over nitrate and nitrite are Excess nitrate is excreted absorbed in the intestine by the kidneys4 ADVANCES in orthomolecular research Volume 3 Issue 10
  7. 7. distant sites in the body?” The answer, it seems, lies in salivary glands is 100-fold greater than in the blood the fact that NO is converted back into nitrite and and that of nitrite it is 1000-fold greater! The rest of nitrate. Although NO lasts only a short time and may the nitrate is eventually excreted in the urine. So both only act directly and locally before it is oxidized into urine and saliva are particularly rich in nitrate and nitrite and then nitrate, this rapid oxidation to both nitrite. It is interesting then why animals lick their nitrite and especially nitrate, allows these much more wounds or that urinating on an injury is considered stable molecules to then travel to distant sites for re- beneficial – the potential benefits of these behaviours conversion back into NO as and when the specific will be discussed in detail in later articles in this tissue requires. In effect, both the nitrite and nitrate act magazine. as pro-drug forms of NO; in other words, nitrate and In summary the opposite biochemical processes of nitrite act as a stable reservoir and a depot for future oxidation and reduction of L-arginine and nitrate NO requirements by all cellular tissues throughout the bioconversion into NO respectively may be viewed as body. In this manner NO can act both locally and at “ying-yang” with the more conventional L-Arginine distant sites in a similar manner to various hormones pathway occurring in conditions when there is plentiful secreted by the thyroid or adrenal glands. of oxygen and the nitrate pathway as a “back-up” An interesting feature of nitrate in the blood is that system occurring in conditions when there is lack of approximately 25% of the nitrate is concentrated back oxygen. The key point is that without this vital back-up in the salivary glands via the entero-salivary circulation system our bodies would not be able to carry out a cycle (See Figure 4). The concentration of nitrate in the number of biological functions. REJUVE NOX REDUCES STRESS AND BOOSTS ENERGY TECHNOLOGYVolume 3 Issue 10 ADVANCES in orthomolecular research 5
  8. 8. DIET Nitrates/Nitrites Higher or Low Oxygen VASODILATOR NO Anti inflammatory Antioxidant Lowers BP Helps arthritis, joint pain & inflammation Diuretic Improves oxygen delivery to the body Gastric Mucosal Protection Benefits conditions like sickle cell disease Prevents Ulcers & Infections6 ADVANCES in orthomolecular research Volume 3 Issue 10
  9. 9. THE ROLE OF NITRATES IN THE BODY Cholesterol Platelets L-Arginine High Oxygen Only Anti-platelet Prevents Clots Prevents Prevents LDL Oxidation Arterial Plaque Prevents Heart Atttacks Anti-bacterial Prevents Prevents Ulcers & Strokes InfectionsVolume 3 Issue 10 ADVANCES in orthomolecular research 7
  10. 10. found to be as effective as a single prescription NITRIC OXIDE therapy for blood pressure! Researchers from Johns Hopkins University reported AND THE an 18% reduction in the 10 year risk of heart disease with the DASH diet. The researchers stated that, “In addition to reducing blood pressure, the DASH diet CARDIOVASCULAR should substantially reduce the risk of coronary heart disease”. SYSTEM Cardiovascular Disease (CVD) which includes high blood pressure (hypertension), angina (ischaemia), arterial damage (atherosclerosis), stroke and heart attack is still the number one cause of morbidity and mortality world-wide. One in three Americans is Similarly, the lower incidence of CVD in patients affected by CVD, and over a billion people worldwide consuming Mediterranean, vegetarian and Japanese are affected by high blood pressure alone. In Canada, diets has been attributed to the high fruit and someone dies from heart attack or stroke every 7 vegetable intake associated with these diets. In fact minutes, with CVD accounting for 30% of all deaths in some researchers have reported that the greatest Canada annually. The costs associated with CVD are protection against CVD is offered by diets with the quite staggering with 2006 cost estimated in the EEC highest content of leafy green vegetables (See Table 2). to be over 200 billion Euros. In Canada, the costs This has led scientists to ask some important questions; associated with CVD in 2009 were estimated at $22.2 “What is the common denominator in such diets?” and billion in physician services, hospital costs, lost wages therefore “What are potential CVD protective and decreased productivity. compounds associated with these diets?” In recognition of these alarming statistics there is a It appears that the riddle has been finally solved. A serious initiative on the part of the government to group of researchers principally from Sweden, the UK increase the daily intake of fruits, vegetables and fiber and the US have independently and collaboratively as well as to encourage people to exercise more. In shown that the nitrate and nitrites in our fruits and North America, the previous recommendation of five vegetables may be the answer and have forwarded servings of fruits and vegetables has been upgraded to convincing evidence to support their hypothesis. a recommended nine servings per day. Are Inorganic Nitrates the Answer? Various epidemiological and cohort studies have Nitrates can be classified into two categories: organic repeatedly and unequivocally reported a lower and inorganic. Organic nitrates are what most of us incidence of CVD with higher intake of fruits and are familiar with; for example glyceryl trinitrate (GTN) vegetables. These findings have culminated in the is what is in the tiny tablets usually dispensed by famed Dietary Approaches to Stop Hypertension pharmacists in glass vials and recommended to be (DASH) study. The DASH diet was specifically designed placed under the tongue for anginal pains. Another to lower blood pressure and recommends eight to ten common organic nitrate compound it isosorbide servings of fruits and vegetables a day, along with dinitrate, which is a more advanced and stable form of consumption of low-fat dairy products. The results of GTN. These organic nitrates have been in use since the the DASH diet in reducing blood pressure have been mid 1800’s. Then there are the inorganic nitrates,8 ADVANCES in orthomolecular research Volume 3 Issue 10
  11. 11. which include potassium nitrate and sodium nitrate. nitrate/nitrite content. As can been seen from Table 2 These inorganic nitrates are commonly found as salt the vegetables with the highest nitrate/nitrite content forms in soils, rocks and plants and have been used are spinach, cabbage and Bok Choy. Other for thousands of years in gun powder, food vegetables such as beet root, collard greens and leeks preservation and for heart ailments by early Chinese also have very high nitrate content. Not only are these physicians. vegetables loaded with nitrate but many of them also A recently discovered 1000 year old Chinese have a high content of the enzyme nitrate reductace manuscript translated by scholars at Cambridge which assists the conversion of nitrate into nitrite, University shows the use of inorganic nitrates by thereby facilitating the conversion of nitrite into NO. Buddhist monks for heart ailments! (See Figure 5) It is This is particularly true for the Eastern herb quite remarkable that the Chinese physicians knew not Cinnamomum. This herb is rarely used by itself for only about the inorganic nitrates but more importantly CVD because it has a fairly low nitrate/nitrite content. they understood their proper use as evidenced by their However, when it is combined with herbs like Dan advice to their patients to “Administer under tongue Shen, which do have high nitrate content, the effect is and have patient swallow the saliva” It is as if the pronounced! In this combination, Dan Shen provides physicians knew back then about the entero-salivary the high nitrate content and Cinnamomum provides circulation of nitrate! the necessary enzyme to reduce nitrate into nitrite and eventually to NO for therapeutic efficacy. Putting under the tongue to cause heart ”qi” to flow freely. Table 2: The nitrate/nitrite content of various widley consumed For treating symptoms such as struck by evil, acute heart pains and cold in the hands and feet western vegetables which can kill a patient in an instant. Look at the patients fingers and those with greenish-black nails are such cases. NITRITE NITRATE Take potassium nitrite (5 measures of a bi spoon) and arsenic sulphide (1 measure of a bi spoon) VEGETABLE CONTENT CONTENT and combine the two into a fine powder. Lift the patients tongue and sprinkle 1 measure (mg/100g) (mg/100g) of a bi spoon under the tongue. If saliva is produced have the patient swallow it. Root Vegetables This is a sure cure. Carrot 0.002 - 0.023 92 - 195 Figure 5. A medical recipe from Buddhist monks at Dunhuang Mustard Leaf 0.012 - 0.064 90 - 95 from medieval times showing that the benefits of nitrates were recognized as early as 900 years ago. (From Lundberg et al., 2008) Green Vegetables Dr. Nathan Bryan and his colleagues have investigated Lettuce 0.008 - 0.215 12.3 - 267.8 the nitrate/nitrite content of various herbs and Spinach 0 - 0.073 23.9 - 387.2 vegetables consumed in the West and by the Eastern cultures. Interestingly, they have found that herbs and Cabbage plants that have been associated with a reduction in CVD are also those with the highest nitrate/nitrite Chinese Cabbage 0 - 0.065 42.9 - 161 content. For example the herb Radix miltiorrhizae, also Bok Choy 0.009 - 0.242 102.3 - 309.8 known as Dan Shen, has a very high nitrate content and is a widely used Traditional Chinese Medicine Cabbage 0 - 0.041 25.9 - 125.0 (TCM) for conditions like angina, myocardial infarction or stroke. Its therapeutic efficacy has been associated Cole 0.364 - 0.535 76.6 - 136.5 with its ability to dramatically raise NO levels. Clinical Melon studies have shown that Dan Shen’s therapeutic benefit is nearly comparable to GTN. Furthermore, Wax Gourd 0.001 - 0.006 35.8 - 68.0 Dan Shen does not produce the tolerance that Cucumber 0 - 0.011 1.2 - 14.3 normally develops with most organic nitrates; inorganic nitrates seem to be free of this restriction. Nightshade Similarly, Bryan’s research group has looked at various Eggplant 0.007 - 0.049 25 - 42.4 commonly consumed western vegetables and theirVolume 3 Issue 10 ADVANCES in orthomolecular research 9
  12. 12. NO and Reduction of Blood Pressure Nitrite blood pressure (mmHg) concentration (μM) Hypertension or high blood pressure is defined as a concentration (μM) 400 Nitrate 0.8 Blood nitrate Systolic Blood Pressure (SBP) of 140 mmHg or higher Systolic blood Blood nitrite pressure or a Diastolic Blood Pressure (DBP) of 90 mmHg or 200 0.4 higher, with normal blood pressure being defined as 120/80 mmHg or lower. However, the risk of negative 0 0 health effects and death is increased in individuals 3 6 12 24 0 whose blood pressure is consistently 115/75 mmHg or TIME (H) Change in systolic higher, with the risk of cardiovascular disease doubling with each increment of 20/10 mmHg above 115/75 -5 mmHg. Figure 6. Association of changes in plasma nitrate and nitrite concentration High blood pressure is associated with an increased -10 with blood pressure. (from Kapil et al., risk of stroke, myocardial infarction (heart attack), 2010) heart failure, kidney failure and cognitive impairment. SBP above 115 is the most important determinant of plasma nitrate levels resulting from consumption of the risk of death world-wide and is responsible for 7- sodium nitrate was accompanied by a reduction in 8 million deaths annually and over 20,000 deaths blood pressure. This suggests that the blood pressure each year in the United States. In Canada more than lowering effect was due to the nitrate being converted one in five people (22.7%) of Canadian adults aged into nitrite and then into NO. Although such changes 20 years and older were living with diagnosed in blood pressure appear relatively small, it must be hypertension. Since approximately 17% of individuals remembered that the dose of nitrate used was not with hypertension are not aware of their condition, the large; equivalent to a single serving of salad true prevalence of hypertension is likely higher. The (approximately a plate or 250 g). Even so, these small statistics in the US are similar, with approximately 22% results could still have meaningful health effects when of adults living with diagnosed hypertension. It has considered over the lifetime of the individuals. For been suggested that in moderately hypertensive instance, over the long term, even a small reduction in patients a 5 mmHg reduction in blood pressure might blood pressure will help reduce the stress put on blood reduce the incidence of stroke by 22 % and coronary vessel walls due to high blood pressure. Additionally, it heart disease by 16 %! is important to consider that the study was conducted The beneficial effects of fruits and vegetables on blood in healthy patients with normal blood pressure levels. pressure are well known. For example, a comparison It is expected that a significantly greater fall in blood of the effect of a traditional Japanese diet (with a high pressure would be observed in hypertensive patients. nitrate content) to that of a diet with low nitrate content Following up on the important findings of the Swedish (as much as 20-times less nitrate) in the same 25 researchers, Dr. Amrita Ahluwalia and her group in subjects (a cross-over study), demonstrated a London conducted an elegant study to prove the point significantly lower DBP by 5 mmHg. that the nitrate rich vegetables can reduce blood Because vegetables are naturally rich in nitrates it pressure via conversion into NO. Again in a double- seems reasonable to investigate whether the blind randomized placebo controlled study, healthy consumption of inorganic nitrate alone, corresponding adult volunteers were given 500 mL of beetroot juice to the amount present in a plate of salad for example, to drink. The amount of nitrate present in the juice was could affect blood pressure in healthy subjects. Dr. Jon three times the amount administered in the Lundberg Lundberg and Dr. Eddie Weitzberg’s group in Sweden study. Three hours after consuming the beetroot juice tested exactly this hypothesis in a small double-blind there were significant reductions in both SBP and DBP placebo controlled, cross-over designed study. Sodium of 10 mmHg and 8 mmHg respectively. The beauty of nitrate in a dose of 7 millimoles of nitrate (see page 2: this study was the clear demonstration of a cause and What is a mole?) per 70 kg adult male was effect relationship between the nitrate/nitrite and the administered for three days after which blood pressure reduction in blood pressure. By monitoring plasma was measured. It was found that DBP was indeed nitrate and nitrite levels as well as blood pressure reduced by a significant 4 mmHg compared to the levels, the researchers were able to show that placebo (sodium chloride with equivalent amount of increased plasma nitrate and nitrite levels sodium). The researchers found that the rise in corresponded directly to a decrease in blood pressure.10 ADVANCES in orthomolecular research Volume 3 Issue 10
  13. 13. There are three noteworthy points that are raised by blocked the NOS enzyme (responsible for NO this study. First, the half to one hour delay that occurs production via the L-Arginine pathway) with specific between the intake of juice and the reduction in blood enzyme inhibitors of the enzyme. As a result of pressure represents the time taken for nitrite to be blocking this enzyme the animals developed converted to nitrate by the bacteria in the mouth and hypertension. This effect was reduced by administering then to be converted into NO in the stomach. Second, nitrites in the drinking water. In a more recent 2011 the study demonstrates a dose response relationship, study, this research group has shown that meaning that the higher the dose the greater the administering nitrates to hypertensive animals (caused effect. Finally, an exquisite relationship was established by removing the kidney) not only reduced blood between raised plasma levels of nitrate/nitrite and pressure but also reduced scarring and other damage blood pressure reduction. For scientists there is no to the tissues in these animals. Furthermore, a much better evidence than a physiological cause-and-effect lower dose of nitrite that did not reduce blood pressure relationship, and this study accomplishes this! still protected against kidney damage, suggesting the A second study by Dr. Ahluwalia’s group provided use of much lower doses of nitrates as a preventive further evidence, and also demonstrated that the measure. More studies are needed in this exciting blood pressure effect they observed could be achieved field. with a lower 250mL intake of beetroot juice. More importantly, they showed that the effects of dietary Other CVD Protective Effects of Nitrates, nitrate from beetroot juice can be sustained over a Nitrites and NO longer period, with the reduced blood pressure effects In addition to their blood pressure lowing effects, lasting for over 24 hours! inorganic nitrates have been shown to have numerous Lundberg and Weitzberg’s group have also used other cardiovascular protective effects including animal hypertensive models to provide further improved endothelial function, improved platelet evidence of the benefits of inorganic nitrates for function, a reduction in the symptoms of metabolic reducing blood pressure. In one of these models they syndrome 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 there is a definite relationship between UV light as a predisposing factor for non-melanoma and melanoma skin cancers especially in light skinned people, there is fair amount of data suggesting that exposure to sunlight can reduce blood pressure as well as offering other health benefits. Previous researchers have attributed these benefits to the formation of vitamin D and melatonin by the skin, however, Dr. Feelisch presents a novel hypothesis to explain this phenomenon. He suggests that there is 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. In fact 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 similar increase in animals has been shown to offer significant cardiac and circulatory protection. In addition, all-cause mortality risk is tightly related to latitude, and seasonal variations in light intensity are correlated with seasonal variations in the incidence of CVD-related events and deaths. Rates of strokes, angina and heart disease are all higher in the winter months than summer. Of course other factors like diet and exercise need also to be taken into account. Dr. Feelisch argues that since nitrate and nitrite levels are under dietary control it could be possible to greatly enhance plasma levels and thereby allow the body to produce NO on demand, where and when the body desires to achieve optimal health benefits.Volume 3 Issue 10 ADVANCES in orthomolecular research 11
  14. 14. Diseased Healthy Vessel Vessel Endothelial Dysfunction Build up of Arterial Plaque Reduced Vessel Elasticity Decreased Blood Flow Decreased Oxygen Delivery Decreased Limb Function Nitrite & NO Improved Endothelial Funtion Increased Vessel Elasticity Increased Vessel Growth Increased Blood Flow Increased Oxygen Delivery Increased Limb Function Figure 7. A comparison of a normal healthy blood vessel and a diseased showing damaged endothelial cells and a build-up of atherosclerotic plaque. The figure shows the beneficial effect of nitrite therapy on diseased blood vessels. (from Patillo et al., 2011). The human circulatory system is the largest organ in cause damage to the fragile endothelial layer. For the body. The health of this enormously important example, high blood pressure results in an excess of system depends on the proper functioning of a specific mechanical sheer forces to be exerted on the layer, type of cell that lines the inside of the blood vessels. which can damage the endothelial cells. On the other These cells are called endothelial cells, and they form hand, tobacco smoke may damage the cells by the lining of every blood vessel in your body, from the causing an excessive generation of free radicals or large aorta to the tiniest capillaries. The endothelial reactive oxygen species. As the name implies these are layer is only one cell thick and plays a vital role in the highly reactive molecules, and they can attack the health of the blood vessel. Any abnormality or endothelial cells, damaging them. Similarly, poor diet damage leads to these cells leads to a condition called in the form of excessive consumption of sugar or endothelial dysfunction, which is thought to be a key cholesterol can initiate endothelial damage. In each initiating factor in the pathology of CVD. case, the net result is the formation of plaques in the High blood pressure, inhaled or ingested toxins (e.g. blood vessels. These plaques are highly dangerous as tobacco smoke), excessive alcohol intake, poor diet they can cause the rupture and release of blood clots, (e.g. excessive cholesterol or sugar) and stress can all resulting in further damage to distant sites and can12 ADVANCES in orthomolecular research Volume 3 Issue 10
  15. 15. also lead to blood vessel blockage - causing heart innate tendency towards “togetherness’, in other attacks or strokes. Once the damage to endothelial words they tend to aggregate or clump together. In cells begins; it is like a chain reaction, with the normal circumstances this is a good thing, since the damage spreading from endothelial cell to endothelial clotting caused by platelets is required to prevent cell. Essentially, one plaque breeds more plaques, excessive blood loss when an injury occurs. However, causing inflammation to spread like wildfire and platelet adhesion, activation and aggregation are also contributing to atherosclerosis of the blood vessel. known to be key factors in the progression of Atherosclerosis is a build-up of plaque inside the atherosclerosis and thus, modulation of platelet arteries, which can thicken and harden over time (see function is an important therapeutic strategy for the Figure 7, next pg). This results in a loss of blood vessel prevention of atherosclerosis. Both beetroot juice and elasticity and flexibility which is critical to how the potassium nitrate have been shown to reduce platelet blood vessel responds to pressure changes. Any blood aggregation. Reduced platelet aggregation or vessel that has reduced elasticity will have increased “stickiness” not only helps to prevent the inappropriate pressure inside it. clumping of cells that can lead to colt formation and Endothelial cells are also important because they are artery blockage, but also allows blood flow to take one of the key sites in the body where NO is place more easily thereby reducing the burden on the synthesized. NO acts as a local signaling molecule heart. and hormone to the neighboring cells, allowing for monitoring of the environment in the vessel and A B C VESSEL DIAMETER ensuring that proper blood pressure and other factors ( nmol/L) 10 10 0.8 0.8 300 300 % CHANGE IN ( mm ) are maintained. NO also has direct actions on the 0.7 0.7 250 250 blood vessel itself. It can travel to the smooth muscle 8 8 0.6 0.6 cells located directly beneath the endothelial cell lining 200 200 0.5 0.5 where it caused vasodilatation or widening of the 6 6 0.4 150 blood vessels. In a situation where excessive free 0.4 150 4 radicals are present, NO is unable to do its job 4 0.3 0.3 100 100 properly since the free radicals will act to “mop” up the 0.2 0.2 2 2 NO, reducing the amount of NO that is able to act to 0.1 0.1 50 50 cause vasodilatation, which is not a good thing! 0 0 00.0 00 Studies using genetic knockout mice (see page 2) that Flow mediated dilation (vessel elasticity) Blood vessel thickness Nitrite levels in the blood lack the endothelial NOS enzyme have shown that Healthy Individuals with these animals are highly prone to cardiovascular Individuals endothelial dysfunction complications. However, nitrite supplementation was able to reduce such endothelial disturbances and help Figure 8. The association between blood nitrite levels and to prevent resulting damage. The Ahluwalia study endothelial dysfunction. Healthy individuals show high nitrite levels discussed above showed a similar beneficial effect in which are associated with high levels of artery elasticity. Individuals humans. They found that beetroot juice effectively with endothelial dysfunction show low nitrite levels, reduced artery reduced endothelial damage as assessed by various elasticity and thicker artery walls. inflammatory markers like C-Reactive Proteins (CRP). Recently, a direct relationship between reduced Metabolic Syndrome endothelial damage and plasma nitrite levels has In 1985, Reaven coined the term Metabolic Syndrome been established in humans (see Figure 8). Overall, to describe a cluster of symptoms that often appear the data points towards a very important role for together and may have a common initiating or nitrate/nitrite in the preservation and support of causative event. This cluster of symptoms include: healthy endothelial function. obesity, hypertension, high lipid/cholesterol levels and diabetes. These deadly conditions are the four Platelet function horsemen of the apocalypse that have targeted one Platelets are cells present in the blood that are distinct quarter of the world’s adult population. from the other two major blood cell types, red and Collectively, these four conditions act to increase the white blood cells. The primary function of the platelets risk of CVD in a synergistic fashion, meaning that the is to aggregate at sites of damage causing clotting. risk increases exponentially rather than additively as Platelets are able to cause this clotting due to their more and more factors become involved.Volume 3 Issue 10 ADVANCES in orthomolecular research 13
  16. 16. Animal studies using knockout mice lacking the NOS enzyme gene (thus preventing NO formation) result in full-blown metabolic syndrome. Various researchers Metabolic Syndrome: have suggested that metabolic syndrome may be due to a deficiency of NO. Addition of nitrate and/or nitrite Are you at risk? Metabolic Syndrome is characterized by the following to the drinking water of these knock-out mice has been collection of symptoms: shown to protect the animals, significantly reduces their lipid and triglyceride levels and also reduces the For Men: • Waist circumference > 40 inches • Triglycerides > 150 mg/dL incidence of diabetes and weight gain. These results • HDL Cholesterol < 40 mg/dL • Blood Pressure > 130/85 mmHg present an intriguing possibility for the prevention and • Fasting Glucose > 100 mg/dL control of metabolic syndrome in humans, although human studies are required to verify these results. For Women: • Waist circumference > 35 inches • Triglycerides > 150 mg/dL Ischemia • HDL Cholesterol < 50 mg/dL • Blood Pressure > 130/85 mmHg • Fasting Glucose > 100 mg/dL Ischemia occurs when there is a lack of oxygen delivery to tissues, often due to partial or complete obstruction of blood flow. For example, endothelial dysfunction leading to atherosclerosis can result in reduced to NO. This production of NO during ischemia. Angina (chest pains), heart attacks and ischemic events is the body’s way of protecting the strokes are examples of ischemic events. When cells and tissues during periods of reduced oxygen ischemia occurs, the tissues that are deprived of blood supply. Studies in mice and rats have shown that an and oxygen suffer irreversible damage which can infusion of nitrite during an ischemic event can result in death. Tissues like the heart, the kidneys and significantly improve recovery and reduce tissue the brain are particularly sensitive to an interruption in damage and death in the heart, lungs, kidney and blood supply. Besides helping to reduce the incidence liver. Other animal studies have also found that of ischemic events in the first place through its ability pretreatment with inorganic nitrate helps to enhance to widen the blood vessels (thereby improving oxygen blood flow to damaged areas following an ischemic delivery to the tissues) and through its improvement of event, which can help to minimize damage. Overall, endothelial function, NO can also have direct effects the evidence points to a preventative and protective in the prevention of ischemic injury. Animal studies effect of nitrates and NO for ischemia. Further studies have shown that during ischemic events, low oxygen in humans will provide further support for the role of acidic conditions result, which causes nitrite to be nitrates in these conditions. CARDIO NOX CardioNOx combines nitric oxide enhancing nitrates with co-enzyme Q10 for superior cardiovascular support TECHNOLOGY14 ADVANCES in orthomolecular research Volume 3 Issue 10
  17. 17. MOVE TO A NEW“BEET!” All the benefits of fruits and vegetables in a convenient tablet Scan to learn more about AOR’s Beets n Berries. Get the mobile app at:
  18. 18. For example, Lundberg and Weitzberg’s group in NITRIC OXIDE , Sweden conducted a small human randomized double-blind-placebo-controlled study, where healthy male athletes were given a dose of 7 millimoles of inorganic nitrate for three days prior to an episode of EXERCISE AND low-intensity exercise on a cycle. This dose of the nitrate corresponded to the amount of nitrate that would be found in a plate of salad. ENDURANCE It is widely recognized that exercise improves a plethora of cardiovascular symptoms including cholesterol, blood pressure, risk of stroke, vascular inflammation, and many others. Furthermore, researchers in the field of Applied Physiology and Sports Medicine have found that exercise also improves the function of the heart, accelerates cardiac rehabilitation, controls congestive heart failure (a condition where the heart is unable to pump the blood The researchers showed that supplementing with even efficiently) and prevents intermittent leg pains caused this low dose of nitrate significantly improved the by poor blood flow due to blockage and hardening of performance of the athletes as measured by amount the arteries. of oxygen being consumed (Vox). In other words, consumption of nitrates reduced the body’s demand Recent research seems to suggest strongly that the for oxygen during exercise. Furthermore, in addition to benefits of exercise on cardiovascular health are in reducing the body’s oxygen requirement, or “oxygen large part due to raised NO levels. Exercise increases cost”, the researchers also noticed that the time taken the production of NO by the endothelial cells. Since to reach exhaustion was also increased. This suggests NO is a potent vasodilator and increased production that consuming nitrates prior to exercise can actually of this molecule causes blood vessels to become reduce and/or prevent exercise fatigue! In a follow-up relaxed and widened, thereby allowing more blood study by the same research group showed that nitrates flow and increased oxygen delivery to the body’s acted in the same positive way when athletes tissues. Lack of exercise, improper diet and lifestyle performed high intensity exercise as well! and ageing will all eventually lead to reduced NO generation at the tissue level, and particularly in the These findings were further supported by the British blood vessels, resulting in a consequent worsening of researchers at Exeter University. Led by Dr. Nigel cardiovascular symptoms. Benjamin, one of the pioneers of the nitrate/nitrite/NO research, this group used beetroot juice instead of Although it is acknowledged that exercise positively sodium nitrate. The effect of beetroot juice was evident influences NO levels one wonders if the reverse is also as quickly as 1 hour after consumption, and reached true. Namely can higher NO levels improve exercise maximum effect after three hours. The studies using capacity? beetroot juice confirmed the findings that nitrates Again researchers at University of Exeter in the UK and reduce oxygen consumption and fatigue during the Karolinska Institute in Sweden have looked into this exercise. A second study by the same British group phenomenon in great detail and have independently showed a similar effect when beetroot juice was taken come up with some surprising findings! Essentially for a longer time period of 15 days. In other words, these researchers have found that raising NO levels the positive effects were still evident when nitrates were can indeed have a positive impact on exercise capacity being consumed on a longer term basis. This was not and endurance! solely an acute phenomenon!16 ADVANCES in orthomolecular research Volume 3 Issue 10
  19. 19. Interestingly, both the British and Swedish studies explain its cell protective effects. At the cellular level, noted that there was no increase in production of lactic NO acts to reduce the generation of reactive oxygen acid in the athletes consuming nitrates! Lactic acid species, which prevents cellular and DNA damage and production normally occurs following exercise and is also helps to prevent apoptosis or cell death. one reason why athletes become fatigued. As oxygen In terms of athletic performance, the potential benefits becomes rate limiting, and tissues start becoming of inorganic nitrates are enormous. For athletes, this starved of oxygen, the body resorts to an alternative, small, but important molecule may open the door for less efficient mechanism of energy production that improved performance without the negative health produces lactic acid as a by-product. However, these and legal implications associated with other sport studies demonstrated that in the presence of NO, enhancing drugs! energy production in the muscles remains efficient, which in turn improves the body’s ability to tolerate high intensity exercise for longer time periods. It has been shown that nitrate supplementation prevents the EXERCISE depletion of a key muscle protein called Increased blood flow and vascular shear phosphocreatine; phosphocreatine measurements are stress due to increased blood pressure an indicator of energy usage in the form of ATP . Research results have suggested that nitrates cause muscle contractions to use less energy. Blood Vessel Skeletal Heart Increased production of Maximal Muscle NO from L-Arginine Mitochondrial Mitochondrial Energy Efficiency Production NO 2.0 150 120 NO2 - 1.5 ATP ( % of placebo ) Increased nitrite storage 90 1.0 60 Heart NO2 - 0.5 30 Oxygen levels decrease as exercise intensity and duration increase 0.0 0 placebo nitrate placebo nitrate In response to Low oxygen levels can ischaemia the cause cardiac ischaemia body mobilizes NO from its stored nitrite Figure 9. Graph showing improvements to mitochondrial function associated with an increased intake of dietary nitrate. Higher NO availability enhanced nitrate levels were shown to improve two key measures of NO mitochondrial function: the efficiency of energy (ATP) production (as measured by oxidative phosphorylation ratio) and also the maximal energy (ATP) production. This means that increasing THE HEART IS dietary nitrates enables mitochondria to produce more energy more efficiently! PROTECTED Nitrite has also been shown to interact directly with the Figure 10. Diagram showing the stress imposed by exercise on mitochondria; the cellular powerhouses or energy tissues like the heart and how nitrate/nitrite can increase NO generating factories of the cells. Mitochondria produce availability and protect the heart tissue when oxygen is limited energy in the form of ATP which is the energy currency , that all cell processes require to function. Dr. Sruti However, the significance of all of these findings Shiva at the University of Pittsburg School of Medicine extends far beyond the field of exercise physiology. For has studied the complex actions and interactions of instance nitrates could be of great benefit to NO at the mitochondrial level and proposes that the individuals suffering from reduced motility or exercise unique exercise enhancing effects of inorganic nitrates capacity. The ability of NO to reduce muscle energy and vegetables rich in nitrates may well be due to their use and extend the time to fatigue could be highly specific interactions with various enzymes involved in beneficial to ambulatory convalescing patients, seniors energy production in the mitochondria. These and heart patients who have a reduced exercise interactions not only explain the exercise enhancing capacity and require assistance in recovery times from and oxygen sparing effects of NO but also help to their illness.Volume 3 Issue 8 10 ADVANCES in orthomolecular research 17
  20. 20. Maintaining healthy colonies of bacterial flora Nitric Oxide and throughout the GIT is essential for good health. Unfortunately, this delicate balance is easily disturbed by poor diet, an unhealthy lifestyle, the use of certain prescription medications like antibiotics and many Gastrointestinal other factors. INTESTINAL MICROFLORA Health 1000 Billion Microorganisms comprising over 500 Different Species Lactobacilli Stomach 1 to 10 thousand The explosion of NO research in recent years has been accompanied by some provocative, brilliant and trend Streptococci Lactobacilli setting findings about this simple molecule. NO plays 100 thousand to 1 million Small a critical role in the health of the gut by protecting Enterobacteria Intestine against various bacterial invaders and by maintaining Enterococcus Faecalis the integrity of the stomach lining, called the gastric Bacteroides 10 thousand to 100 million Bifidobacteria mucosa. The gastric mucosa is a layer of mucous that Peptococcus lines the inside of the stomach, and it essential for Peptostreptococcus Large Ruminococcus Intestine protecting the cells of the stomach, and the rest of the Clostridia (colon) 10 billion to 10 trillion body, from the acidic environment within. Lactobacilli The human gastrointestinal tract (GIT) is a long Figure 11. The bacteria or “microflora” of your gastrointestinal winding tube consisting of various types of cells from tract. A healthy balance of these microorganisms is essential for the mucosal cells in the mouth to the epithelial cells of maintaining good health. the intestines and everything in between. Proper functioning of the GIT is crucial for the maintenance of In the mouth, saliva plays a critical role in not only optimal health. However, the GIT and its cells must lubricating the food to facilitate swallowing but also in constantly cope with a complex assortment of friendly the production of key enzymes that begin the digestive and pathogenic bacteria, yeasts, toxins, chemicals, process. Humans produce approximately 1 liter of enzymes and nutrients like fats, carbohydrates, saliva per day. This salvia is produced in the mouth proteins and fiber. Further adding to the enormous and eventually makes its way into the stomach. complexity of this task is the fact that all of this must Nitrates obtained from the diet (for example from the take place across a wide range of pH levels, from consumption of lettuce, beetroot, spinach, leeks etc.) acidic, to neutral to alkaline. are rapidly converted into nitrite by a specific type of bacteria housed on the surface of the tongue. Spitting or not swallowing the saliva following consumption of Gut Microflora and NO the above nitrate rich foods results in a lower nitrite In terms of cell number, bacterial cells in the GIT concentrations in the blood; hence the age-old but outnumber human cells by a staggering 9 times! These sensible advice, “eat your greens but don’t spit”. bacterial cells encompass many different species and Similarly, the use of mouthwash also results in lower have a wide range of functions – some of them friendly blood nitrite levels due to the destruction of the and some of them detrimental. For example, some bacteria on the tongue. It is therefore advisable not to bacteria in the gut are responsible for the synthesis of use mouthwash immediately after eating vegetables, certain essential nutrients like B vitamins, vitamin K so as not to negate the benefits of these nitrate rich and folic acid, others are important for facilitating the foods. A better strategy would be to drink a cup of absorption of nutrients like calcium, and still others green tea after consuming a plate of salad or spinach; produce harmful toxins. Real estate in the GIT is highly this is a healthy alternative that will not destroy these sought after, resulting in intense competition between important nitrate reducing bacteria. It is also important all the different bacteria for nutrients and space. The to note that the conversion of nitrates into nitrite is two key sites with the highest density of the bacterial enhanced by the presence of certain natural cells are the mouth and the large intestines. compounds like vitamin C, and antioxidants like the18 ADVANCES in orthomolecular research Volume 3 Issue 10