Nitric Oxide, Superoxide & Peroxynitrite

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Nitric Oxide
     Nitric Oxide Synthesis
     Physiological (Beneficial) Pathways of Nitric Oxide
     Pathological (Detrimental) Pathways of Nitric Oxide
     Causes of Elevated Nitric Oxide Levels
           Prolonged or Repetitive Immune Activation
           Hyper-Excitement of NMDA Receptors
           Hyper-Excitement of Vanilloid Receptors
           Hyper-Excitment of Muscarnic Acetylcholine Receptors
           Stress or Psychological Trauma
           Physical Trauma
           Ionising Radiation Exposure
           Pre-Existing Autoimmune Disease or Inflammatory Condition
           Genetic Predisposition to Elevated Nitric Oxide Levels
           Low Tissue Oxygenation and Capillary Budding
           Psychological Stress or Trauma
     Too Low Nitric Oxide Levels? David Whitlock's Hypothesis
     Formation of Superoxide
     Genetic Predisposition to Elevated Superoxide Levels
     Superoxide Dismutase (SOD)
     Formation of Peoxynitrite
     Other Reactive Nitration Species (ROS) formed from Peroxynitrite
     Pathological (Detrimental) Pathways of Peroxynitrite
     Antioxidant Protection against Peroxynitrite
Balance between Free Radicals and Antioxidants
Dr Martin Pall's Peroxynitrite Protocol
     Preventative Measures
     Treatment Protocol
Dr Paul Cheney's Peroxynitrite Protocol
     Methods of reducing elevated Peroxynitrite levels
     Methods of Reducing Elevated Peroxynitrite & Superoxide Levels
     Methods of Blocking Nitric Oxide Production
     Other Methods
     General Comments on Dr Paul Cheney's Hypothesis on Peroxynitrite
Other Protocols
     Dr Jacob Teitelbaum's Peroxynitrite Protocol
     Dr Gareth Nicolson's Peroxynitrite Protocol
     Dr Neboysa Petrovic's Peroxynitrite Protocol
Markers and Tests for Peroxynitrite


Dr Martin Pall has hypothesised in his book 'Explaining "Unexplained Illnesses": Disease Paradigm for Chronic Fatigue Syndrome, Multiple Chemical Sensitivity, Fibromyalgia, Post-Traumatic Stress Disorder, Gulf War Syndrome, and Others' (2009) that the NO/ONOO- (Nitric Oxide) cycle may be a significant factor in some cases of CFS, FM, Multiple Chemical Sensitivities (MCS) (see the Effects of Toxicity page for more information), Post Traumatic Stress Disorder (PTSD) and perhaps even Autism. He suggests that the elevated Nitric Oxide and Peroxynitrite levels in these conditions is a shared root cause or factor, which is also comorbid in a large number of other well-accepted diseases, for example viral related illnesses, allergies and autoimmune conditions. This might also explain how some sufferers of one of these conditions may develop multiple instances of other inflammatory conditions concurrently, as they share a root driver. Autoimmune diseases such as Lupus and Rheumatoid Arthritis, are reported to have elevated iNOS activityand peroxynitrite, NF-kB and cytokine elevation at the sites of the autoimmune-related inflammation. These factors shall be examined in this article.

In these illnesses, short term stressors such as viral or bacterial infection, physical or psychological trauma or exposure to various toxic chemicals are thought to raise the Nitric Oxide (oxidant) levels in the body, exaccerbating their symptoms. The elevated Nitric Oxide levels react with Superoxide in the body, a byproduct of a number of bodily processes, to form the very harmful rogue oxidative species Peroxynitrite. The formation of Peroxynitrite causes a wide variety of oxidative damage to the body, particularly to mitochondrial enzymes, membranes and also hemoglobin, as well as destroying the protective antioxidant enzyme Superoxide Dismutase (SOD) (and other mechanisms for stimulating Superoxide production), thereby allowing Superoxide levels to build up, causing more of the Nitric Oxide to react with this Superoxide, thereby perpetuating or worsening the condition by producing more Peroxynitrite.

Raised Superoxide levels through physical or mental overexertion can also trigger increased inflammation by reacting with Nitric Oxide as well as burning up our mitochondrial enzymes and membranes, impairing mitochondrial function. The increased inflammation also makes it harder for the mitochondria to repair effectively, creating slow recovery periods from overexertion, even when the patient rests for days or weeks at a time.

Pall argues that therapy should focus on down-regulating the NO/ONOO- cycle biochemistry rather than on treating symptoms. He has recommended that nutritional/mineral support and individual and full-spectrum antioxidant preparations that together may assist in down-regulating this NO/ONOO- cycle mechanism are to be considered a sensible approach as part of an overall treatment programme.

The intensity of the Nitric Oxide and Peroxynitrite cycle does appear to vary in the patients of the above cases, but it does appear to be a factor in the overall illness of each respective person to some degree. It may be a primary driver or cause in one patient, and play a secondary part compared to other primary causes, e.g. heavy metal toxicity, in the next patient. In some phases of an illness, inflammation can play a smaller part secondary to other factors, and in other phases it may play the dominant role. Symptoms of inflammation vary, depending on where exactly in the body the inflammation is and what the exact nature of it is. Nervous system aberrations and cognitive disability ('brain fog') often point to excessive inflammation and/or excitotoxicity in the brain.

Some summaries of Martin Pall's Peroxynitrite hypothesis can be found at the links below. The final link is probably the most technical and comprehensive read, which is presumably a summary of Pall's book. I recommend purchasing Pall's book of course..

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Nitric Oxide (NO):

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Nitric Oxide, a.k.a. Nitrogen Monoxide or NO, is a important signalling molecule in mammals, including humans. Most other signalling molecules in the body are non-gaseous, which makes NO quite unusual as it is a gas at room or body temperature. It is both an oxidising and reducing agent, depending on what molecules or ions it is reacting with. In other words it is both a free radical (pro-oxidant) and antioxidant.

NO has a very short half life of around 1 second, and only exists for a few seconds. Nitric Oxide (NO) is not the same as Nitrous Oxide (N2O), a general anaesthetic and fuel for dragsters. Another oxide form of Nitrogen is Nitrogen Dioxide (NO2).

NO is involved in both physiological and pathological processes, depending on its concentration and precise conditions. It is thus anti-inflammatory and pro-inflammatory. These two properties of NO are examined below.

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Synthesis of Nitric Oxide:

NO is synthesised by the reduction of the amino acid Arginine and O2 by various Nitric Oxide Synthase (NOS) enzymes and also by the reduction of inorganic nitrate.
There are three types of Nitric Oxide Synthase (NOS enzymes):

Both nNOS and eNOS are Calcium dependent, producing no NO unless Ca2+ is present. The cytoplasm of these cells typically have low levels of Ca2+. Cellular Calcium levels are tightly regulated. However, any pathway that increases cellular/cytoplasmic Calcium levels will also increase NO production and NO levels. Also, any pathway that induces iNOS, under immune system inflammatory conditions, will also increase NO production and NO levels.

As the nNOS and eNOS enzymes in the cells are not fully saturated/utilised at any one time in Nitric Oxide and Citrulline production from L-Arginine, then increasing the L-Arginine concentration in the cell above normal will likely increase the rate of NO production. Thus it is inadvisable for individuals with elevated NO levels to consume L-Arginine rich foods or take L-Arginine supplements to push their L-Arginine levels above normal as it will likely worsen one of the root causes of their condition.

Tetrahydrobiopterin (BH4) is one of the cofactors involved in NO production. BH4 is a form of reduced Biopterin and is also an protective mechanism against the free radicals produced by cellular inflammation (i.e. Neopterin). BH4 has other uses in the body and is also used as a cofactor in enzymes involved with the production of other neurotransmitters and precursors, besides NO, including 5-HTP, L-Tyrosine and L-DOPA. Neopterin, BH4 and Biopterin are discussed on the Immunity page in the Pterins section.

When a cellular NOS enzymes have limited BH4 or L-Arginine, they may produce Superoxide (O2-) instead of NO. L-Arginine levels tend to be relatively low (sub-normal) in those with elevated NO and/or peroynitrite levels, as their L-Arginine body pools have been used up in NO production. Superoxide has most of the same properties of NO as a messenger molecule. Producing Superoxide outside of the relative safety of the inner mitochondrial membrane may result in the NO/ONOO- cycle being exacerbated, i.e. the NO present in the cell reacting with the Superoxide to form the harmful Peroxynitrite (ONOO-).

BH4, in its role as antioxidative protection inside a cell reacts with Peroxynitrite and is oxidised to Biopterin. Oh course, BH4 can and is also oxidised by Neopterin, an inflammatory cytokine, which may or may not itself be stimulated by the presence of Peroxynitrite - a viral or other infection, other sources of free radicals, chemical sensitivity, and/or an allergic/intolerance response may also be responsible potentially. BH4 and Biopterin levels can be measured as discussed below. It is likely that this is one pathway for Peroxynitrite formation in those with excessive levels.

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Physiological (Beneficial) Pathways of Nitric Oxide: In low concentrations, NO is a beneficial messenger molecule in the body. Appropriate NO levels are important in protecting organs such as the liver from ischemic damage or ischemia, that is, damage caused by a restriction in blood supply. NO is also used as an antimicrobial agent by certain types of White Blood Cells.

'Nitric oxide (NO) protects the heart, stimulates the brain, kills bacteria, helps prevent blood clots that are the cause of most heart attacks and strokes, enhances oxygen delivery to tissues, and helps regulate blood pressure and blood flow to different organs. It is present in most living creatures and made by many different types of cells. It was a sensation when it was discovered that this simple, common air pollutant,-which is formed when nitrogen burns, for instance in automobile exhaust fumes-could exert so many important and life saving functions in our body.'

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Pathological (Detrimental) Pathways of Nitric Oxide:

Some of the pathological effects of elevated NO radicals are listed below. There are other mechanisms of elevated NO levels but they are not fully understood at this time.

Chronic expression of NO is associated with various carcinomas (cancers of the epithelial cells) and inflammatory conditions such as juvenile diabetes, Multiple Sclerosis (MS), Arthritis and Ulcerative Colitis (according to a 1997 study).

Causes of Elevated NO Levels:

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Too Low Nitric Oxide Levels? David Whitlock's Hypothesis:

'An Engineering Perspective on CFS' (7 Nov. 2008) - by Dave Whitlock

In the above article (discussed by me on the Mitochondrial page), Dave Whitlock argues that low basal NO levels may explain low levels of mitochondrial regeneration, resulting in lower numbers of mitochondria per cell than a normal, healthy person. NO (Nitric Oxide) is a major regulator of ATP levels. Low NO levels causes low ATP levels, which thus disables autophagy, preventing recycling of mitochondria. There is more peroxynitrite damage observed not because peroxynitrite levels are high and NO levels are higher, but because there is less recycling of mitochondria occuring (less autophagy) and hence less repair of peoxynitrite-damaged proteins and lipids. In other words, there is a resulting accumulation of peroxynitrite-damaged proteins. Because of low NO levels, there is less synchronisation between cells in terms of their energy output (in a muscle group or particular organ), meaning some are overloaded and some are underloaded. According to Whitlock, techniques do not exist to measure if adjacent cells are working 'in sync'. Whitlock proposes a number of methods of boosting NO levels (or more specifically NO donors) in the body to allow the body to produce more mitochondria, which include (in no particular order and not necessarily recommended by me as this is a THEORY) taking Nitroglycerine, L-arginine, Viagra, eating more green leafy vegetables, and meditation.

Paul Cheney and Martin Pall argue the exact opposite, that NO levels and Peroxynitrite levels in CFS patients tend to be higher than normal, rather than lower, on account of the enzymatic activities associated with over-immune system activation, on account of prolonged exposure to viri or bacterial infections etc., amongst other factors. Cheney proposes a number of methods of reducing one's NO production. As to who is correct, I am not certain, and it presumably depends on the exact individual in question as to what is going on on a specific biochemical level and where. Everyone however is probably in agreement that poor mitochondrial function is behind cardiac insufficiency.

Supplements are available that are designed to stimulate eNOS production, to improve vascular function in afflicated individuals (who do not produce sufficient enthelial NO). This can help to prevent atherosclerosis on account of arterial wall thickening, and also improve oxygen and nutrient delivery. NO stimulating supplements are also taken by weight lifters to improve performance. Clearly those who are producing too much NO should not consider such a regime as it will exacerbate their symptoms. One example of such a supplement is Xymogen's N.O.max ER, which contains Arginine alpha-ketoglutarate and ACTINOS2 Whey Peptide Fraction, but shown to stimulate Endothelial NO production. Such a supplement could probably be used for those travelling at high altitudes or with erectile problems (if NO is really the main cause).

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Superoxide is a relative unreactive free radical (Gerdes, 2003). Superoxide can also act as a intracellular messenger molecule, like NO.

There are two main types of Superoxide. The predominant form of Superoxide is OO- or O2-, where there is an unpaired electron (characteristic of free radicals). Superoxide does not generally move very far from its point of creation within the cell and cannot easily pass through cellular membranes on account of its negative electrical charge. The alternate form is its acidic form, where it binds with a Hydrogen (H+) ion (from an acid) to form HOO. HOO has no negative charge and is more easily able to pass through cell membranes. However, it is only makes up 0.1% of the body's total Superoxide.

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Formation of Superoxide:

There are 5 main pathways to the creation of Superoxide. These are listed in descending order (by Pall) of the amount of Superoxide the relative pathways produce on average:

In the following 2007 paper, Alvarez et al. identify NADPH oxidase enzyme activity as being the main source of Superoxide, the vasoconstrictor, (the inflammatory response from Phagocytes), which reduces the amount of NO available from increased iNOS activity than there would otherwise be, to form Peroxynitrite.

'Role of NADPH oxidase and iNOS in vasoconstrictor responses of vessels from hypertensive and normotensive rats'. Alvarez et al. 2007.

'Hypertension is associated with elevated levels of circulating proinflammatory cytokines, which may alter the vascular expression of enzymes like inducible nitric oxide synthase (iNOS) and modify the regulation of vascular tone during this pathology. Indeed, increased vascular iNOS activity and/or protein expression have been described in hypertension. The role of iNOS-derived NO in vasoconstrictor and endothelium-dependent vasodilator responses has been previously analysed by our group and others in lipopolysaccharide or interleukin-1-beta-stimulated arteries. However, the participation of iNOS-derived NO in vasoconstrictor responses in unstimulated vessels is not well studied.

Oxidative stress can affect vascular reactivity by different mechanisms. Reactive oxygen species function as second messengers, activating numerous signalling molecules and play an important role in vascular physiopathology. Several sources of superoxide anion (O2-) within vessels have been described. Among them, xanthine oxidase, uncoupled NOS and COX can produce O2- in different conditions. However, at the vascular level it is well established that nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase, present in all three vessel layers, is the main source of O2-. An increase of O2- production has been observed in human and different experimental models of hypertension, including spontaneously hypertensive rats (SHR). More specifically, the enhanced O2- generation in hypertension is a known result of the activation of vascular NAD(P)H oxidase.

The mechanisms whereby increased O2- production might contribute to high blood pressure are currently under active investigation. However, it is also well known that by interacting with NO, O2- forms peroxynitrite, thus decreasing NO availability for smooth muscle relaxation. Hypertension is associated with changes in vascular responses, such as impairment of endothelium-dependent vasodilator responses or enhancement of vasoconstrictor response to different agonists. Several studies have analysed the relationship between increased O2- production and the impairment of endothelium-dependent relaxation in hypertension. However, the O2- contribution to the altered vasoconstrictor responses in hypertension as well as its relationship with the iNOS-derived NO is less studied. The present study was performed to analyse how hypertension might alter the role of O2- in the vasoconstrictor responses to phenylephrine, the sources of this O2- and its relationship with iNOS-derived NO.'

Hypertension is associated with increased activity and/or expression of iNOS as well as increased production of O2- in different vascular beds; these changes might contribute to the alterations in vascular tone occurring in this pathology. The main results of the present study suggest that the increased production of O2- derived from NAD(P)H oxidase, observed in aorta from hypertensive rats, counteracts the enhanced production of NO derived from iNOS, occurring in hypertension, and the modulation exerted by NO of vasoconstrictor responses.'

'...hypertension increases iNOS expression but decreases the bioavailability and the modulation elicited by iNOS-derived NO of contractile responses in aorta as a result of the increased O2¥? production from NAD(P)H oxidase.'

Increased Superoxide formation tends to result in increased formation of the rogue oxidant molecule Peroxynitrite (which degrades into various Reactive Nitrogen Species (RNS)). Peroxynitrite is a vasoconstrictor, in a similar way to Superoxide and is implicated in cases of Hypertension.

'Peroxynitrite versus nitric oxide in early diabetes'. Robert D.ÊHoeldtke et al. 2003.

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Genetic Predisposition to Elevated Superoxide Production:

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Superoxide Dismutase:

The majority of Superoxide is produced during respiration, inside the mitochondria, but there are mechanisms in place to lower its levels on account of it being sufficiently damaging to cellular structures.

The endogenous antioxidant enzyme that is responsible for neutralising Superoxide free radicals are known as Superoxide Dismutase (SOD). There are 3 main types of Superoxide Dismutase enyzymes, each found in a specific location. Each type of SOD is encoded by its own gene and each has a distinct structure.

The SOD antioxidant enzyme is effectively oxidised by Superoxide, which reduces the Superoxide. The SOD can then be recycled by reduction so it can be used again to neutralise the next Superoxide free radical. However, if any Superoxide reacts with NO to form Peroxynitrite, this Peroxynitrite can actually destroy the SOD enzyme. Excessive destruction of SOD means fewer protective antioxidant enzymes to keep O2- at bay, so more O2- builds up. In the presence of elevated NO levels, yet further Peroxynitrite can be produced. Mitochondrial damage caused by excessive Superoxide levels in one mechanism of many in some CFS cases.

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Peroxynitrite (ONOO-):

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The Peroxynitrite anion is shown above. Peroxynitrite is an unstable 'valence isomer' of the nitrate ion, NO3-. It is mostly commonly written as 'ONOO-' but can also be expressed as 'ONO2-'.It is a powerful oxidant/oxidising agent (not actually a free radical) and nitrating agent. It is also a vasocontrictor (see Superoxide section above for details).

Most of the harmful effects of Peroxynitrite are produced not by the ONOO- itself but by its breakdown products.

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Formation of Peroxynitrite:

Peroxynitrite is formed by the reaction of two free radicals, Nitric Oxide with Superoxide.

O2- + NO. -> ONO2-

The two free radicals combine so readily on account of the presence of an unpaired electon on the outer shell of each. The resulting molecule, Peroxynitrite, is not a free radical but a powerful oxidant. The reaction is said to be diffusion limited. In other words, every time a molecule of O2- collides with an NO molecule, the react to form ONOO-.

Superoxide is an oxidising free radical, as is Nitric Oxide, but Peroxynitrite is more powerful an oxidant than the sum of its constituent parts.

Dr Paul Cheney, as stated in some of his seminars, believes that Peroxynitrite is primarily formed by Superoxide (a byproduct of ADP to ATP conversion, i.e. energy production, inside each cell) leaking out of the mitochondria and reacting with Nitric Oxide (NO - a byproduct of NOS enzyme activity). This is not strictly speaking correct. Some ONOO- is indeed formed inside the mitochondrial membranes, but normally only a very small amount. The Superoxide Dismutase (SOD) present in the mitochondria inhibits ONOO- production by reacting with the O2- before it can react with NO to form ONOO-. The mitochondria also contain some levels of Glutathione (hopefully sufficient) to protect the mitochondrial membranes against ONOO-. As discussed above, Superoxide is produced outside of the mitochondria as well inside them, so the Superoxide that reacts with NO to form ONOO- in the cytoplasm of cells and also outside of the cells themselves is far more likely to be responsible for cystoplasmic and extracellular ONOO- than Superoxide 'leaking' out of the mitochondria.

The Superoxide produced as part of respiration and ADP to ATP conversion stays inside the inner mitochondrial membrane. It can only escape and 'leak out' if the mitochondrial membrane is damaged. This can and does of course occur, especially in some CFS patients with excessive free radical damage, but not to the extent that would be necessary to account for such Peroxynitrite build up in the body. Indeed, this degree of mitochondrial membrane damage would likely result in death, as demonstrated by laboratory mice that had no mitochondrial SOD and had their mitochondrial membranes attacked and ravaged by O2-, which did not live very long. In the majority of CFS cases, this route is unlikely to be the dominant one for ONOO- production in the body. The mechanisms cited by Martin Pall above seems to make more sense, including the production of SOD by cytoplasmic NOS enzymes (outside the mitochondria) in the absence of sufficient L-Arginine or BH4 to make their usual NO.

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Other Reactive Nitrogen Species (RNS) Formed from Peroxynitrite:

Reactive Nitrogen Species (RNS) are a family of antimicrobial molecules and free radical species derived from NO. RNS react together with Reactive Oxygen Species (ROS) (e.g. Superoxide) to damage cells, causing nitrosative stress. RNS and ROS are collectively referred to as ROS/RNS. ONOO- is a highly reactive oxidising and nitrating agent and tends to react with other molecules to produce additional types of RNS free radicals and oxides of Nitrogen (e.g. NO2). NO2 is a good oxidiser also, and which can also react with NO to produce N2O3, another powerful oxidiser. Examples are listed below.

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Pathological (Detrimental) Pathways of Peroxynitrite:

Excessive peroxynitrite formation is postulated in the 2004 Paul Cheney interview document (see the Cardiac Insufficiency page for more information) as the primary force behind free radical damage and impaired mitochondrial function.

Peroxynitrite (free radical) formation is partly behind the development of Cancer and also Coronary Artery Disease. Cheney believes this is the main driver behind CICM and CFS.

Antioxidant Protection Against Peroxynitrite:

Antioxidant molecules that react and neutralise the Carbonate Radicals and Nitrogen Dioxide radicals formed from Peroxynitrite, rather than Peroxynitrite itself directly, can also be termed Peroxynitrite Scavengers.

Dr Paul Cheney stresses the importance of Glutathione and Selenium (an antioxidant metal and constituent of Glutathione) as a defence against ONOO- and in this he is correct, as Glutathione is the body's main intracellular and extracellular antioxidant enzyme, particularly with regards to controlling free radical damage. However, whilst both Glutathione and SOD help to prevent oxidative damage to the mitochondrial membranes, SOD is directly responsible for containing Superoxide, which is arguably of most primarly importance, Glutathione being a secondary line of defence.

The nutritional element Selenium and the mitochondrial cofactors CoEnyzme Q10 and Lipoic Acid also offer a degree of protection. Selenium is an antioxidant metal and forms part of the Glutathione enzyme. The mitochondrial cofactors can help to neutralise ONOO- and also recycle Glutathione.

However, in the presence of Mercury, Selenium is not able to bind with Glutathione Peroxidase, and so it is not able to assist SOD in breaking down Superoxide. In addition, most CFS patients (if not all) do not produce enough Glutathione on account on methylation problems. Inadequate levels of Glutathione can result in intra- and extra-cellular oxidative damage.

Superoxide Dismutase (SOD) is the protective antioxidant enzyme against free radical damage caused by Superoxide produced inside the mitochondrial membranes. Whilst not protection against ONOO- per se, it does prevent the formation of ONOO- inside the mitochondria by reacting with the Superoxide (O2-) formed, so that it cannot combine with any Nitric Oxide produced inside the mitochondria. Of course, most of the ONOO- produced is probably formed outside of the mitochondria, as discussed above.

The presence of external antioxidants, i.e. those present in antioxidant rich foods that are ingested, will also help to minimise oxidative stress cause by elevated Peroxynitrite levels to some degree, but the internal antioxidants, i.e. those produced by the body, are of primary importance. Dr Martin Pall argues that these are not generally that effective as single agents in preventing ONOO- mediated damage to cells and tissues, but that combinations of them may be somewhat more effective overall.

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Balance between Free Radicals and Antioxidants:

It is easy to think of Free Radicals as essentially bad, and Antioxidants as essentially good. This is not quite true. Free radicals are inadvertently produced during respiration inside our mitochondria, as part of the electron transport chain, in the form of Superoxide (O2-) when O2 accidentally accepts an electron. There will always be some Superoxide formation during respiration. The electron transport chain is a balance of redox reactions, i.e. transfer of electrons from one molecule (oxidising it) to another (reducing the recipient). This is a finely balanced process. The body of course has to try to prevent free radical damage from the Superoxide free radicals as much as it can, and it does it mainly by the SOD enzyme and Glutathione and other antioxidants. Antioxidants protect against free radical and oxidative stress by being oxidised themselves, and are thus often classed as reducing agents. The levels of these enzymes are generally matched to the normal expected output of these Superoxide radicals. The body does not produce too many more antioxidants than it needs as some of these (the reducing agents) would create reductive stress and inhibit redox reactions in the body, which are essential to life. The electron transport chain in mitochondria would not function with too much reductive stress. Denham Harman MD PhD, the creator of the Free Radical Theory of Ageing in 1954, stated that there are dangers with high antioxidant intake as too much can leave one feeling very weak (i.e. inhibiting mitochondrial function). I myself have observed this phenomenon.

Nitric Oxide is a signalling molecule used in the nervous system and also is a vasodilator in the vascular system. Antioxidants help to reduce bad cholesterol (LDL) to good cholesterol (HDL), helping to reduce atherosclerotic plaque formation, and limit free radical and oxidative damage of the vascular tissue (preventing hardening) but some antioxidants have been linked to increased risk of atherosclerotic plaque formation (e.g Vitamin C)! High levels of certain antioxidants have been linked to increased risks of Coronary Heart Failure, e.g. Vitamin D and E. Most antioxidants are vasodilators, including the polyphenols OPCs and Resveratrol, but some in excess may possibly cause vasoconstriction if there is an inhibiting of NO in the vascular tissue.

iNOS is an important biological enzyme, and the production of free radicals is an important part of our immune system, which are used to kill invading microbes etc. iNOS is not simply the 'bad guy'. However, it is when iNOS is excessively activated that excessive free radical production occurs and the inflammatory vicious cycle of Peroxynitrite derivative formation occurs. Too much antioxidant intake can result in the suppression of the immune system and render one more susceptible to infections and disease. This has been noted in a a 1970s study of antioxidant therapy. Excessive antioxidant intake is also a risk factor for Type II Diabetes and also interfere with some Cancer treatments which rely on cancer cells killing themselves prematurely with internal free radicals.

Antioxidants also have a pro-oxidant role in the body, in that if they reduce an anion (e.g. Ascorbic acid reduces a metal anion such as Fe3+ to Fe2+), then that reduced Cation can then be oxidised by Hydrogen Peroxide, to form Fe3+ again but also an OH. radical. Excessive supplementation with antioxidants can thus ironically result in more free radical formation, which is the opposite of what we are trying to achieve.

Certain antioxidant sources also contain compounds that suppress mineral absorption. These are 'anti-nutrients' and include oxalic acid from cocoa, tea, spinach and berries, phytic acid (lipoic acid) from whole grains and mazie, and tannins in tea. Oxalic acid and tannins will also increase the chance of kidney stone formation, whereas Phytic (Lipoic) acid actually reduces it. Phytic acid is also a useful mobilising agent for heavy metals if used correctly. These are however only some of the sources of antioxidants and there are many others - which may also become problematic in other ways in too high dosages.

Antioxidants themselves can be beneficial in the right doses but toxic or damaging in too high dosages. This is true of Vitamins A, C and E, and minerals like Selenium, and likely a number of other plant sourced antioxidants. One can mitigate this of course by varying the sources of one's antioxidants and not relying solely on one type of antioxidant, but there is still the issue of reductive stress and pro-oxidant formation even with a varied source of antioxidants if one consumes too many. Some argue that it is best to obtain all one's antioxidants from food sources, rather than rely on supplemental forms. Clearly antioxidant supplements can be very useful, but they should be taken in appropriate dosages and not simply eaten like sweets around the clock. The Nitric Oxide/Peroxynitrite therapy discussed below involves taking antioxidants which are targetted to optimal Peroxynitrite scavenging, and to a lesser extent Nitric Oxide. Clearly this is for those with excessive levels of inflammation, rather than those with normal levels of Nitric Oxide. However, even with these antioxidants, there is an optimal amount for a given individual, and taking any more than this is not improving treatment but may be counterproductive. A medicine can become a poison depending on the dosage.

In addition to considerations regarding the quantities of antioxidants to take, one should also consider the types to take and in what form. From a muscle testing perspective, the body may be best able to reduce inflammation with certain types of antioxidants than others, and some of the antioxidants mentioned on this page may not be optimal for your body at a given point in time. To get the best results, it helps to provide the body with the optimal combination and in just the right quantities.

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Dr Martin Pall's Peroxynitrite Protocol:

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Preventative Measures:

Pall had made the following suggestions to avoid exacerbating the NO/ONOO- cycle. These include the avoidance of:

Excessive physical or mental exercise is probably one of the biggest contributors to exacerbation of inflammation.

I would also add the following things to avoid:

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Treatment Protocol:

Dr Martin Pall has suggested a number of means of reducing and managing Peroxynitrite formation. These include taking a variety of useful antioxidants, B-vitamins, amino acids, Essential Fatty Acids, minerals (specifically Magnesium), and reduced Glutathione and its precursors. Dr Paul Cheney's protocol is actually based upon many of Pall's recommendations, but he adds a few of his own, as you would expect.

Please find below the NO/ONOO- protocol suggested by Dr Martin Pall to Dr Grace Ziem in 2007, who subsequently made a few additions, namely nebulised, inaheld reduced-gluatathione and hydroxocobalamin. This is still correct as far as I am aware at the time of writing in 2009, but as you would expect it will be subject to revision and amendments in the future.

Please note that I have referenced all of the above supplements elsewhere on this site as being beneficial for a wide variety of roles and applications. Of course, this approach should not necessarily be prescriptive.

Some of the forms of delivery of some of the items, e.g. Glutathione and Hydroxocobalamin). There may also be minor differences in delivery mechanisms of some of the supplements, e.g. Glutathione and B12 nasal spray as opposed to capsules or sublingual tablets or liquid. It might be optimal to ascertain using a variety of nutritional-based tests in addition to muscle testing which of these the body actually requires and in what form. To address the variety of most critical problems in the body, whatever they may be, excessive ONOO- or otherwise. A treatment protocol therefore could include some of the items below but others that are addressing other needed areas.

I have some comments regarding superior forms of the compounds listed below that he would personally substitute, as there is no reason to use or recommend the inferior form, and some additional items.

A number of supplements developed by Allergy Research Group / Nutricology in conjunction with Martin Pall, or with Pall in mind, are listed below. These are described by Nutricology as follows.

'These individual and full-spectrum antioxidant formulations were developed by Martin Pall, Ph.D., and Stephen Levine, Ph.D., to provide nutritional assistance in down-regulating the NO/ONOO- cycle mechanism.'

- FlaviNOx (contains extracts of milk thistle seed, bilberry leaves, ginkgo leaves, grape seed, green tea, cranberry juice and hawthorn).
- FibroBoost (mentioned above)
- Super EPA
- MVM-A - antioxidant, vitamin and mineral formula
- CoQ-Gamma E - a combined CoQ10 and Gamma E supplement.

It should be noted that whilst Hawthorn may be useful for many patients, for cardiac support and as an antioxidant, it is a mobiliser of heavy metals. If you have high levels of circulating heavy metals in your body, then taking anything with Hawthorn in it may well make you very ill and actually increase levels of Peroxynitrite-based inflammation rather than decrease it. This is in my opinion a rather large oversight for such a product.

There are many supplements by other manufacturers that may work as well if not better with a given individual, but these are a convenient range. e.g. Jarrow Formulas' Resveratrol Synergy is a convenient mixture of Grape Seed OPCs, Grape Skin Extract, Green Tea Extract and Tiger Cane Root Extract.

Pall in his book 'Explaining "Unexplained Illnesses"' also mentions the following supplements and drugs with reference to NO/ONOO- treatment, but which do not appear explicitly in the Pall/Ziem treatment protocol. These are listed below. Some of these items I have referenced elsewhere on this site as being beneficial for a wide variety of applications. Pall has mentioned one or two items, namely drugs and antibiotics, that I would not necessarily recommend and comments with follow shortly.

Experimental or drug-related therapies mentioned by Pall are listed below. These are not necessarily recommended by me and if they are considered, then the above should be tried first in any case, or used in conjunction with drug therapies, rather than relying on drug therapies alone.

Two notable and extremely important omissions from Pall's suggestions and exploratory discussions are the neurotransmitter GABA and the amino acid L-Glycine. Both are inhibitors and will help to lower excitotoxicity in the neurological system, to redress the balance between excitatory amino acids/neurotransmitters Glutamate and Aspartate vs the inhibitory amino acids/neurotransmitters Taurine, Glycine and GABA. GABA can be directly supplemented if deficient, either as GABA or PharmaGABA. L-Glycine can be supplemented in its free form.

In addition to the above, I would also recommend looking into the original triggers or causes of Peroxynitrite formation and any other factors that are interfering with your healthy immune system function, neurotransmitter balance and mitochondrial function. This could include assisting the body to detoxify heavy metals from the body (a major source of free radicals in the body), assisting the body to fight any infections that may be present (using adaptogenic herbs or otherwise), ensuring health neurotransmitter and hormone function (through adaptogenic stimulation, nutrient supplementation and/or actual hormone/prehormone/neurotransmitter supplementation and detoxification of heavy metals if present). It may also include dietary changes including eliminating any foods that are causing inflammation in the GI tract. And finally addressing any skeletal and stress issues. If the mitochondrial membranes are damaged as well as clogged up with toxins, one might want to consider a Phospholipid Therapy and Far Infrared Sauna programme, in order to restore healthy mitochondrial function (at least on the membrane level). If the NO/ONOO- cycle is impairing your mitochondrial function, in the short term, then you want to do all you can to boost the function as much as possible to assist in the recovery process, until the mitochondrial enzymes etc. have been regenerated in the absence of excessive oxidants. This should all be considered in conjunction with the direct methods for lowering NO and ONOO- levels as wells as the mitochondrial assistance discussed above.

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Dr Paul Cheney's Peroxynitrite Protocol:

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Methods of reducing elevated Peroxynitrite levels:

Dr Paul Cheney (in 2004) has suggested a number of means of fighting high Peroxynitrite levels.

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Methods of reducing elevated Peroxynitrite and Superoxide levels:

Cheney (in 2004) has suggested a number of means of fighting high Peroxynitrite and Superoxide levels.

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Methods of blocking Nitric Oxide Production:

Cheney (in 2004) has suggested a number of means of blocking Nitric Oxide production.

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Other Methods:

In Cheney's 2006 seminar, he additionally recommends the following treatments, specifically relating to Peroxynitrite and Nitric Oxide management.

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General Comments on Dr Paul Cheney's Cardiac Hypothesis Specifically Relating to Peroxynitrite:

I am doubtful that Peroxynitrite always the main factor in bringing on impaired mitochondrial function, as Paul Cheney believes, but believes there are other equally significant causes. However, it is likely that the NO/ONOO- cycle is implicated in some capacity. It could just be that the way Dr Cheney has presented the information, he has heavily emphasised some areas heavily almost arbitrarily at different points in time. It is difficult to know what is still part of his protocol when it is not mentioned each time - it could be that he likes to emphasise the newest developments he is working on or it could be that he has dropped a certain methodology. There are some Cheney followers on the internet who attend his annual seminars and summarise their key points, and they find it very hard to follow just exactly what he is saying and relating it to what he said before. I am not completely convinced by the heavy focus on peroxynitrite in 2004 (although he does believe it is relevant), which seems to have dropped or been put into perspective in his 2006 seminar. There also seems to be some confusion over the respective roles of peroxynitrite in preventing hemoglobin from saturating with oxygen and the observed tendency for hemoglobin in CFS cases from his clinic in being very slow to desaturate. Which is the primary driver in low hemoglobin oxygen levels? It could just be that Cheney has a tendency to overemphasise any particular point he is discussing or illustrating at the expense of others, and depending on what he is talking about, one will arrive at a significantly different understanding of CFS in general. As stated above, the main precepts of his theory could be largely explained also simply by general, blanket reduced efficiency in all systems caused by mitochondrial function, toxic overload and immune system overload.

Cheney has himself stated that mitochondrial inefficiency is behind the reduced cardiac function or capability. The bottle neck here is not the cardiac function but the mitochondrial capability and the efficiency of the biochemical steps and enzyme production associated with the Krebs cycle in the mitochondria. So perhaps the heart is not really protecting the body but is merely responding to the circumstances it finds itself in, i.e. doing as much as it can given its mitochondrial efficiency. How much of this adaptation is phenotype related and how much is merely responding to a reduced mitochondrial capability based on specific causes such as toxicity and insufficient nutrient levels, pH and so on? If the bottle neck was the heart, and improving mitochondrial capacity of the body as a whole put more strain on the heart, i.e. the heart was the problem not the mitochondrial dysfunction, then decreased metabolism would in a sense be a protective mechanism for the heart. But it isn't, so in a sense, Cheney's whole argument is contradictory. If one argues that an actual non-mitochondrial related problem is to blame for its insufficiency, then Cheney would be right about the 'protective mechanism'. This is explored more below.

Whilst the body is reputedly 'protecting itself' by slowing down its metabolism and pumping less blood around the body, even if more oxygen was being supplied to the tissues, this would not mean that metabolism would be raised TOO much, as the Krebs Cycle would still be flawed and not working properly. However, raising metabolism globally (i.e. improving mitochondrial function) would be a good thing as it would not put a strain on the heart as the heart would have more energy to operate closer to normal in a healthy manner. If any extra 'strain' was put on the heart would of course depend on what tissues were increasing their metabolism relative to the others (i.e. if the heart was improving its metabolism more than the other tissues, or the other way around).

Perhaps then the argument as to why the body is trying to keep metabolism lower mainly resides on impaired glutathione production and other antioxidant availability in the body, as increasing metabolism increases the level of superoxide production, which can damage the mitochondrial inner and outer membranes as well as the mitochondrial DNA; and also the chance of superoxide leaking out out of the mitochondria (and creating Peroxynitrite), and this causing damage to the outer membrane and to proteins and cell membranes thoughtout the body. However, increased metabolism should result in an increase in glutathione production and cofactor production - is this always the case? It depends on the availability of ATP and what the ATP is being allocated to - to increase energy output or to produce more glutathione and SOD. Of course, glutathione production is often a problem in CFS patients, but lowering metabolism doesn't necessarily prevent Peroxynitrite formation for the previously stated reasons. The only way the body is going to produce more glutathione is through increased metabolism (more energy) in conjunction with increased levels of the relevant nutrients. The argument that the body is protecting itself from oxidative stress by lowering metabolism/mitochondrial efficiency is also in doubt, as stated below as increased antioxidant intake can alleviate and even eliminate signs of inflammation from oxidative stress, but still the body 'chooses' to keep metabolism low. However, as Cheney stated, perhaps this is down to phenotype adaptation.

A discussion of Paul Cheney's ideas about breathing techniques and Peroxynitrite are discussed on the Oxygenation page.

Cheney argues that one should not place one's focus on fighting off bacterial, fungal or parasite infections as these organisms thrive in the low oxygen environment of a CFS patient, which he argues is that way to protect the heart (which I do not personally buy). Cheney argues that if one corrects mitochondrial function and the NO/ONOO- problem, then higher O2 levels will be a result and the respective infections will 'sort themselves out'. Of course, viral infections are not dependent on oxygen as they are not alive - as long as the host is alive, they can spread. All these infections play on a weak immune system or at least one that is preoccupied with the cycle of inflammation in the body. Seeing as these infections are actually stressors or instigators of NO/Peroxynitrite production, then it would follow that in order to effectively lower NO production, one has to lower the immune responses in the body (i.e. the inflammation), and this would presumably mean helping the body to fight off these infections so the immune system has a chance to downregulate itself to normal levels of activity. This goes for all the instigators and root causes of the NO/ONOO- cycle. It is no good staying stressed as it will not help.

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Other Protocols:

A number of other specialist researchers and doctors in the field of CFS and ME have put forward their own treatment protocols for elevated peroxynitrite levels. These include Dr Jacob Teitelbaum, Dr Garth Nicolson, Dr Neboysa (Nash) Petrovic as well as Martin Pall, PhD himself.

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Dr Jacob Teitelbaum's Peroxynitrite Protocol:

Dr Teitelbaum's NO/ONOO- Protocol, described a being a series of nutritional and mitochondrial energy treatments, is summarised below.

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Dr Gareth Nicolson's Peroxynitrite Protocol:

Dr Gareth Nicolson's Peroxynitrite Protocol is summarised below. Nicolson was involved in trials of the mitochondrial support / repair supplement call NT Factor, that includes mitochondrial cofactors and phospholipids.

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Dr Neboysa Petrovic's Peroxynitrite Protocol:

Dr Neboysa Petrovic's Peroxynitrite Protocol is summarised below.

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Markers for Elevated Peroxynitrite Levels:

As far as I am aware, there is no direct measurement available to detect for Nitric Oxide or Peroxynitrite. In a live patient, NO, O2- and ONOO- are continuously being produced - NO is a consultative but short-lived neurotransmitter. Peroxynitrite and Supeoxide, when produced, will tend to react with certain types of groups on proteins and also lipids that it comes across. If one was to take a sample of blood from a patient, by the time the sample got to the laboratory, all of the Peroxynitrite would have reacted and oxidised the respective bodily tissues and compounds. However, a number of indirect measurements can be made. Peroxynitrite is a nitrating and oxidising agent, so it is possible to measure either directly or indirectly the effects of these two activities.

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