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..
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.
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):
Neuronal NOS (nNOS) - is found in fairly constant levels (according to cell type) in many neurons in the brain, spinal cord and peripheral nervous system; and also in many other types of cells in the body.
Endothelial NOS (eNOS) - is found in fairly constant levels (according to cell type) in the endothelial cells that line the blood vessels; and also in many other types of cells in the body. In this role, eNOS acts as a vasodilator.
Inducible NOS (iNOS) - is found only in very small quantities unless it is induced, typically under inflammatory (oxidative) conditions (e.g. shock, toxicity or infection). iNOS also plays a role in modulating vasoconstrictor responses in hypertension. iNOS induction is considered to be part of the emergency pathway of NO.
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.
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.'
Role as a Messenger Molecule (Neurotransmitter) [nNOS]:
NO is highly reactive - as well as being an oxidising agent, it can also act as a reducing agent, reacting with O2, Chlorine, Bromine and other species. It diffuses well across cell membranes. However, it has a chemical lifetime of only a few seconds at the most. These attributes make it ideal as a inter- and intra-cellular signalling molecule (neurotransmitter).
Nitrosylation and Other Protein Conversion Applications:
NO is also involved in the process of Nitrosylation, i.e. the addition of a Nitroysl (N=O) group to a protein. This is an important biological application of NO. Thus it hels to convert thiol groups (S-H), including Cysteine residues in proteins, to form s-nitrosothiols (RSNOs). S-Nitrosylation is an important mechanism for dynamic, post-translational regulation of most or all major classes of protein. NO is involved in a number of other protein conversion pathways.
Role as Vasodilator and Vascular Regulator [eNOS]:
NO is one of the main Endothelium-Derived Relaxing Factors (EDRF). These are factors that are released by the endothelium (interior cell wall lining of blood vessels) to help induce smooth vascular (blood vessel) muscle relaxation and increased blood flow. In other words one of its functions is as a vasodilator. Vasdilators enlarge the blood vessels, increase blood flow and reduce blood pressure.
Nitric Oxide activates the heterodimeric enzyme soluble guanylyl cyclase by binding to it. Guanylyl Cyclase is known as the Nitric Oxide Receptor. Activated Guanylyl Cyclase enables the production of cyclic GMP (cGMP). Cyclic GMP in turn activates and regulates the enzyme G-kinase. G-kinase modifies proteins in the cell through phosphorylation, including myosin light chain phosphatase, resulting in the deactivation of myosin light-chain kinase, and subsequently the dephosphorylation of the myosin light chain, causing smooth muscle relaxation.
NO production tends to be elevated in those individuals who live at altitude, as its vasodilation properties (when generated by eNOS) help to increase blood flow and hence increased chance of oxygenation in the lungs, thus preventing hypoxia. NO is also involved in the production of male erections through its vasodilation properties. Erectile dysfunction may result from too much vasoconstriction in the penis. Viagra (Sildenafil citrate) stimulates erections by enhancing signalling through the NO pathway in the penis (cGMP generation). Nitroglycerin and Amyl Nitrate serve as vasodilators (esp. of the sphinctre) because they are converted to NO in the body.
NO contributes to blood vessel homeostasis by inhibiting vascular smooth muscle contraction (vasoconstriction) and growth, platelet aggrgation and leukocyte (white blood cell) adhesion to the endothelium (blood vessel lining). This is why impaired NO pathways are often noted in individuals with atherosclerosis, diabetes and hypotension (elevated blood pressure). A high salt (NaCl) intake has been shown to attenuate NO production but without controlling bioavailability.
Role as Antimicrobial Agent in the Immune System [iNOS]:
NO is generated as part of the normal immune system response by White Blood Cells known as Phagocytes (that ingest their targets) - including monocytes, macrophages and neutrophils. Phagocytes have both inducible and inhibitory enzymes to control NO. NO secreted as an immune system response uses NO's free radical properties and it is toxic to bacteria, causing the bacteria DNA damage and degradation of their iron sulphur centres. Many pathogenic bacteria have however developed NO resistance. Inflammatory cytokines are also active in inducing iNOS.
Phagocytes also consume O2 and use it to generate O2-, which is used by the NADPH oxidaze enzyme to generate a variety of different oxidants that are used to kill the fungi and bacteria that they ingest. This can be a substantial source of O2- and other oxidants in infected tissues. An increase in O2- production may thus result from the immune system's inflammatory response.
Reactive Nitrogen Species (RNS) is a family of antimicrobial molecules derived from NO produced via the enzymatic activity of inducible Nitric Oxide Synthase 2 (NOS2) gene. NOS is expressed in the liver by macrophages (WBCs) and is inducible by a combination of lipopolysaccharide (a.k.a. LPS - found on the outer membrane of gram-negative bacteria, acting as endotoxins) and certain cytokines.
As stated above the NO produced by the iNOS inside Phagocytes is an emergency pathway of the body, active during times of infection and shock etc. In this role, the NO acts to modulate the vasoconstriction response of the blood vessels to Superoxide production (also from the Phagocytes), producing Peroxynitrite. iNOS is known to cause some increased vascular permeability. Increased iNOS activity is associated with hypertension, which is itself often a product of increased Superoxide production. Please see the Superoxide Synthesis section for more information.
'Evaluation of iNOS-dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide.' Fujii et al. 2000.
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).
The direct toxicity of endogenous NO is considered to be modest. However, its toxicity level increases greatly when it reacts with Superoxide (O2-), with which it readily and rapidly combines. The higher the levels of NO present, the greater the risk of it reacting with Superoxide to form Peroxynitrite.
NO is also an oxidising free radical in its own right, and causes some oxidative stress on the body. In high concentrations, NO is a harmful oxidising agent that can cause excessive oxidative stress on the body's membranes and tissues. Sustained levels of NO production can result in direct tissue toxicity. However, the majority of the pathology of elevated NO is through Peroxynitrite formation.
As mentioned above, NO can protect organs such as the liver during Ischemia (damage caused by the restriction of blood supply to an organ). . NO can however contribute to reperfusion injury, a type of cellular injury occurs when blood returns to an area that had previously had its blood supply constricted, by increasing inflammation and oxidative damage. In such circumstances, NO reacts with the respiration intermediary product Superoxide to form the damaging oxidant molecule Peroxynitrite. Peroxynitrite by contrast is significantly more toxic than NO.
'Nitric oxide forms complexes with transition metal ions, including those regularly found in metalloproteins. The main trap for NO is oxyhemoglobin, which binds NO faster by five to six orders of magnitude than oxygen. The reaction with haemoglobin produces nitrate and methaemoglobin (met-Hb)...Other NO-sensitive metalloproteins are NOS, cytochrome P450 (22), ferritin, ceruloplasmin, myoglobin, cyclo-oxygenase, catalase, ribonucleotide reductase and several components of the mitochondrial respiratory chain. These reactions have wide implications for the physiologic and toxic effects of NO.'
As NO levels in the body become elevated, NO increasinly oxidises Hemolglobin. In a healthy individual, approximately 1% of total Hemoglobin has been oxidised (mainly by NO). However, in individuals suffering from oxidative stress, on account of increased NO levels, the Methemoglobin levels may be significantly higher. The oxidation of Hemoglobin (Haemoglobin) to Methemoglobin, a form that cannot bind with O2 is discussed on the Tissue Oxygenation and CFS page. Other hemo-proteins are also discussed on that page in the Porphyrins and Heme section.
Inhibition of Mitochondrial Enzymes:
NO can inhibit (oxidise) the last enzyme in the electron transport chain of mitochondrial function, known as Cytochrome C Oxidase or Complex IV.
Both NO and Peroxynitrite can oxidise one of the Iron-Sulphur protein-based enzymes in the Krebs cycle (Citric Acid cycle), known as Aconitase. This is discussed below in the Peroxynitrite section.
Both of these two enzymes are important for mitochondrial function and the generation and transport of ATP (i.e. energy production) inside every cell in the body. Pall argues that the Aconitase enzyme has a lower activity in CFS patients, and perhaps this is why, because of excessive NO/ONOO- levels.
Formation of Dinitrogen Trioxide:
NO also reacts with Nitrogen Dioxide (NO2), presumably in lieu of reaction with Superoxide, to form Dinitrogen Trioxide (N2O3). N2O3 is another type of Reactive Nitrogen Species (RNS). This reaction is reversible. All nitrogen oxides are good oxidising agents.
NO + NO2 = N2O3
Excessive Cyclic GMP Formation:
Excessive cyclic GMP formation also plays a part in the pathological path for NO.
Vascular Collapse in Septic Shock
Elevated NO levels can contribute to vascular (blood vessel) collapse associated with septic shock.
As mentioned in the introduction to this page, elevated NO can arise by a number of pathways. The most relevant to CFS, ME and Fibromyalgia patients is probably the infection route, that upregulates NO, production. According to Pall, in CFS and ME patients this is mostly commonly viral infection(s) and bacterial infection(s), less commonly protozoan infections such as toxoplasmosis. In Fibromyalgia patients, viral infections are most common stressors, with bacterial infections usually playing a secondary role if relevant.
Over successive infections, NO production may become increasingly elevated, of course mediated by a large number of other biochemical and psychological factors. This is perhaps why infections are a major trigger in causing CFS, as NO levels in an already impaired body with lowered Glutathione levels and elevated toxicity levels, can result in excessive Peroxynitrite formation. Some treatments for elevated NO and ONOO- levels may thus be geared towards eradicating any remnants of the triggering infection or any new infections or cumulative infections, and also boosting and taming the immune system, perhaps in conjunction with avoiding anything that causing excessive inflammation, such as problem foods.
Raised Neopterin levels, a pro-inflammatory and oxidative cytokine, as well as TNF-alpha, may lead to Inducible Nitric Oxide Synthase (iNOS) gene expression and Nitric Oxide synthesis, based on studies in rat vascular smooth muscle cells (Hoffmann et al., 1998).
L-Glutamate (or Glutamic acid) is the most important excitatory neurotransmitter in the brain and plays an important part in brain chemistry. It is released by many different types of neurons and stimulates other neurons at the synapses. The excitatory neurotransmitters act to stimulate the next neuron (i.e. postsynaptic neuron) to fire an electrical impulse.
Inhibitory neurotransmitters tend to inhibit firing of neurons. The most important of these is GABA, which is synthesised from Glutamine in the presence of Active Vitamin B6 (P5P). GABA is examined more on the Adrenal and Endocrine System page. Taurine is another, which performs many vital functions in the body, including nutritional metal transport into the cells. Taurine is examined in more detail on the Nutritional Deficiencies page. The third main inhibitory neurotransmitter is the amino acid L-Glycine.
GABA and Glutamate levels are balanced in the brain both in terms of absolute concentrations and relative ratios in a healthy individual. A P5P deficiency can thus result in an elevated Glutamate to GABA ratio in the brain and elevated levels of excitotoxicity. Excitotoxicity is the term used when the levels of the excitatory neurotransmitters are too high, at which point the level of neuronal activation or induced firing of neurons become neurologically damaging. Excitotoxins, specifically free glutamate, and the neurological damage they can cause, are examined on the Excitotoxins section on the Nutritional page, with respect to Glutamate and MSG.
N-methyl-D-aspartate (NMDA) receptors in the nervous system system are found in various nerve cells or neurons in the brain, spinal cord and peripheral nervous system. They are also found in some non-neuronal cells. They are stimulated by the excitatory neurotransmitters,i.e. the amino acid Glutamate (Glutamic acid) and Aspartate (Aspartic acid). There are four main classes of receptor that are stimulated by excitatory neurotransmitters. These are the NMDA receptors, AMPA receptors, kainate receptors and metabotropic receptors. The NMDA receptors are of interest here in their role in Nitric Acid production. Whilst the NMDA receptor was thus named because it is the only receptor to repond to NMDA (a synthetic, non-naturally occuring excitatory substance), this is not the biological function of these receptors (as NMDA is not actually produced in the body). The primary biological function of NMDA receptors is to respond to Glutamate and Aspartate. NMDA receptors are also stimulated by the amino acid Glycine and by polyamines. There are in fact 4 structurally distinct types of NMDA receptor, rather than one generic type. Aspartate fulfils other functions than acting as an excitatory neurotransmitters and it is involved in the Krebs cycle for energy production in the mitochondria.
Dr Martin Pall has stated that a state of hyper-excitement of the postsynaptic cellular NMDA receptors by the presence of elevated Glutamate and Aspartate levels (i.e. excitotoxicity) and/or insufficient GABA, Taurine and/or Glycine levels, results in the receptor opening up a channel in the cell membrane, and allowing an influx of Calcium (Ca2+) ions and an egress of Potassium (K+) ions. NMDA receptor over-activation can thus also result in depleted postsynaptic cellular Potassium levels. The influx of Calcium ions into the cells stimulates the nNOS and eNOS Nitric Oxide Synthase enzymes, leading to increased Nitric Oxide production in these postsynaptic cells. Pall suggests that a second Glutamate receptor, e.g. AMPA or kainate receptor, adjacent to the DNMA receptor, may indirectly act to increase the sensitivity of the NMDA receptor to stimulation. nNOS is usually found in muc high concentrations in neurons than eNOS, so that most of the NO production from NMDA receptor activation in such cells is thought to be due to nNOS.
Pall suggests NMDA receptors may perhaps increase Peroxynitrite levels directly, although there is at present no evidence to suggest this, the mechanism for elevated Peroxynitrite being a consequence of elevated NO levels in the presence of presumably elevated Superoxide levels.
Pall is uncertain as to whether NMDA stimulation by Glycine or polyamines plays any role in multi-system illnesses and the NO/ONOO- cycle.
Pall speculates that the documented mechanism of NO (in the learning and memory process) to act as a retrograde messenger in the brain, increasing the long-term potentiation of receptors, diffusing from the postsynaptic cell to the presynaptic cell and next acting to stimulate the release of Glutamate may also act as another potential factor in NMDA receptor overstimulation, as it may create a vicious circle of further NO production and Glutamate release.
Pall also speculates that Peroxynitrite's effect on lowered mitochondrial function (discussed below) including lowered ATP production may have a knock on effect on the NMDA receptors, which become more hypersensitive to stimulation when ATP levels are low. The absence of ATP creates a lowered electrical potential across the outer (plasma) membrane of the cell, which causes the Magnesium (Mg2+) ions to diffuse much more easily from the NMDA receptor site, resulting in these NMDA receptor sites being much more sensitive to stimulation by Glutamate and Aspartate. This may add to the vicious circle of the NO/ONOO- cycle.
The role of NMDA overactivation in the NO/ONOO- cycle may of course vary between patients of CFS, Fibromyalgia, MCS and GWS etc, as does the role of the NO/ONOO- cycle. Pall believes that it is an important trigger mechanism in (the perpetuation of) these conditions. In MCS, Fibromyalgia and CFS, there are studies reporting improved symptoms in the chronic phase in response to NMDA antagonists. Magnesium, an NMDA antagonist, is well documented as being useful in the treatment of these conditions, and is frequently chronically deficient in patients of such conditions.
According to Pall, NMDA receptor stimulation is documented to cause an increase in iNOS activity, NF-kB activity and the synthesis of inflammatory cytokines in the immune system, although it is unclear whether this is a direct mechanism or simply a result of increased NO production (which could equally derive from other NO generating causes).
Pall states that excessive NMDA receptor stimulation is known to play important roles in several neurodegenerative disorders including Parkinson's Disease, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease) and AIDS-related Dementia. Brain damage involving NMDA receptor overstimulation has been found in a number of the most important diseases of the brain. Pall suggests that a drug for treating Alzheimer's Disease, called memantine, a known inhibitor (antagonist) of NMDA receptors, is additional evidence of this important role that NMDA receptors play in the condition.
Pall has suggested that the toxicity stressors of Mercury, Hydrogen Sulfide and/or Carbon Monoxide poisoning play a part in the intiation of Multiple Chemical Sensitivies (MCS), and that their toxic responses on the body are greatly lowered by NMDA antagonists, implying that these toxins render NMDA receptors to be more sensitive or to be overstimulated. The original stimulation of the NMDA receptor may have been a toxin (in particular organic solvents), a psychological event, or even the party drug Ketamine, but once the pattern of increased NO production and NMDA stimulation is established, then even if the original stressor is removed, the pattern has taken on a life of its own and requires other methods of intervention to break of the cycle.
An article 'Fibromyalgia, Excessive Nitric Oxide/Peroxynitrite and Excessive NMDA Activity' by Martin Pall can be read at the link below. NMDA receptor over-activation, as well as other aspects of the NO/ONOO- cycle are examined in more detail in Pall's book.
'The NMDA receptor (NMDAR) is an ionotropic receptor for glutamate (NMDA (N-methyl D-aspartate) is a name of its selective specific agonist). Activation of NMDA receptors results in the opening of an ion channel that is nonselective to cations. This allows flow of Na+ and small amounts of Ca2+ ions into the cell and K+ out of the cell.'
'NMDA (N-methyl-D-aspartic acid) is an amino acid derivative acting as a specific agonist at the NMDA receptor, and therefore mimics the action of the neurotransmitter glutamate on that receptor. In contrast to glutamate, NMDA binds to and regulates the above receptor only, but not other glutamate receptors. NMDA is a water-soluble synthetic substance that is NOT normally found in biological tissue. It was first synthesized in 1960s. NMDA is an excitotoxin; this trait has applications in behavioral neuroscience research.'
As discussed on the Adrenal page, studies on mice have revealed that the heavy metal Mercury increases the release of endogenous glutamate, inhibit glutamate uptake, reduce mitochondrial activity, and decrease ATP levels.
Magnesium supplementation/availability helps to counteract the influx of Ca2+ ions from NMDA stimulation and thus lower the amount of NO released by NMDA receptor stimulation. Zinc also helps to make the NMDA receptor polarisation more difficult.
The algae Spirulina is often recommended by practitioners as a good source of protein, nutrients and Vitamin B12 as well as being an excellent Peroxynitrite scavenger. There is a potential downside in that it is relative high in Aspartic Acid and Glutamic Acid. However, if taken in moderation, the overall effect relative to the NO/ONOO- cycle will be a beneficial one rather than a detrimental one. Please see the Nutritional Deficiencies page for more information.
The Vanilloid Receptors in the body respond to vanilloids as well as acidic pH, heat etc. Vanilloids are compounds that contain a functional vanillyl group. Examples of vanilloids include vanillin, vanillic acid, homovanillic acid, Vanillylmandelic acid (VMA) and capsaicin.
Natural vanilla flavour contains a large number of different compounds (incuding vanillin), and synthetic forms in food contain just laboratory synthesised vanillin.
VMA is a metabolite of Vitamin B2 and B3 metabolism, as well as the adrenaline and noradrenaline pathway. Excessive levels of the 'stress' neurotransmitters adrenaline and/or noradrenaline (norepinephrine) levels over a period of time may result in elevated VMA levels - high VMA levels may thus be an indicator of this. Please see the Hormone and Neurotransmitter Dysfunction page for more information on VMA and Homovanillic acid.
Capsaicin is the 'heat' in hot chilli peppers and other capsicum peppers.
According to Dr Julius Anderson, when the Vanilloid receptor is stimulated, it opens up channels in the cell's plasma membrane, which allows Calcium ions to flow into the cell. Thus influx of Ca2+ ions increases Nitric Oxide synthesis. Vanilloid receptor stimulation has been demonstrated to lead to NMDA receptor stimulation. Many vanilloid-receptor-containing neurons release glutamate as their neurotransmitter, leading in turn to stimulation of NMDA receptors in the postsynaptic neuron.
Pall believes that the overstimulation and overactivity of the vanilloid receptors is a factor in Multiple Chemical Sensitivities (MCS) cases. Excessive vanilloid activity has also been documented in Fibromyalgia patients.
The consumption of hot or spicy foods is generally associated with increased blood circulation and vasodilation, and this may be partly owing to increased NO production, amongst other factors. Hot components of foods are discussed with relation to potential imbalances they can cause on the Digestive Disorders page. All of the above vanilloids mentioned above that are found in food sources, e.g. Vanilla, Capiscum etc. are hot in nature. It should be noted that depending on the patient's exact condition, warming herbs and foods (containing Salicylates) may be useful in resolving dampness issues, according to TCM and Ayurvedic Medicine, and may help to stimulate digestion and blood circulation - but as a side effect may temporarily elevate vanilloid receptor stimulation and inflammation. However, one cannot sometimes fix all problems at once, and sometimes one may need to make temporary 'sacrifices' in the interests of achieving one's goals sequentially and indeed achieving the overall goal of wellness.
Natural vanilla does have some health benefits that should be mentioned in this context, including antioxidant properties, anti-cancer properties, as an aid to preventing the sickling in red blood cells in Sickle Cell Disease as well as a treatment for stomach ulcers and insomnia (sedative properties).
If over-stimulation of Vanilloid Receptors is indeed a problem in your case (as well as excessive NO/ONOO- in general), unless you are eating large quantities of Vanilla flavoured products every day, it should not be an issue. Elevated Adrenaline and Noradrenaline levels may well be more of an issue. Consumption of black pepper and other spicy food may be ill advised.
Hyper-Excitment of Muscarnic Acetylcholine Receptors:
Muscarinic receptors (mAChRs) are G protein-coupled Acetylcholine (ACh) receptors found on the outer plasma membranes of certain neurons and other cells. One of their roles is acting as the main end-receptor stimulated by the neurotransmitter ACh released from postganglionic fibres in the parasympathetic nervous system. Their name derives from their sensitivity to muscarine compared with nicotene (which is not a biological function of the receptors in the body). Their counterparts are nicotinic Acetylcholine receptors (nAChRs). Both these classes of receptor are thus stimulated by ACh.
There are 5 types of mAChR receptors, M1 to M5. M1, M3 and M5 (M-odd receptors) are quite similar and when stimulated, produce increased levels of cytoplasmic Calcium ions (Ca2+) in the cell. M-odd receptors differ from NMDA and Vanilloid receptors in that they release their Ca2+ by a different pathway. They trigger synthesis of the chemical messenger IP3 (Inositol Triphosphate - a Phytate), which releases intracellular Ca2+ from certain Ca2+ stores that are sequestered away in the cell (rather than simply allowing outside Ca2+ into the cell as the other receptors discussed above do). The increased intracellular Ca2+ concentration will lead to increased NO production by nNOS and eNOS (the two Ca2+ dependent NOSes).
Pall suggests that excessive ACh levels and over-stimulation of the Muscarinic receptors on account of exposure to pesticide organophosphate or carbamate pesticides and/or organic solvent toxicity are triggers found in some MCS and CFS cases. In insects and invertebrates, these pesticides kill by inhibiting the enzyme Acetylcholinesterase, theenzyme that breaks down Acetylcholine. This allows ACh levels to build up, thus causing toxicity through excessive Muscarinic receptor activity. This releases more NO, thus feeding the NO/ONOO- cycle. A similar pattern is expected in human cases of exposure to such chemicals.
Excessive DMAE or Centrophenoxine supplementation may result in elevated ACh levels and potentially hyper-excitement of the Muscarinic ACh receptors, thereby increasing ONOO- inflammation.
Low Tissue Oxygenation and Capillary Budding:
Low levels of tissue oxygenation is likely to be a significant factor in many CFS cases. When the target cells at the capillaries are not receiving enough oxygen over a period of time, perhaps because of clogged up capillaries, high levels of oxidised hemoglobin (methemoglobin) or low 2,3-BPG levels, they will start to release a chemical called Vascular Endothelial Growth Factor (VEGF). This is a chemical signal to produce more capillaries in that area, or capillary budding as it is sometimes referred to. The idea is to produce more capillaries locally which should hopefully increase blood supply to those affected cells. The endothelial linings of these newly created 'budded' capillaries produce extra nitric oxide (NO), in order to induce vasodilation and increase blood supply to these target cells. If the extent of capillary budding is significant, then there may be significantly elevated levels of NO in the body.
Hypoxia can also result in increased Superoxide production, which may react with the additional NO being produced in similar conditions and produce more Peroxynitrite.
Pall has suggested that exposure to the following toxic compounds (cumulative effect over long term, low level exposure (or one or more) or a single high-level exposure incident - or consecutive incidents) are implicated in the initiation of the illnesses of CFS, ME, Multiple Chemical Sensitivies, Fibromyalgia by stimulating the Nitric Oxide/Peroxynitrite cycle:
Organophosphorus Pesticides - CFS, ME, MCS - one of most common causative factors
Carmbamate Pesticides - MCS
Organochlorine Pesticides - MCS
Pyrethroid Insecticides (Commercial and Domestic) - MCS
Volatile Organic Solvent Exposure - MCS - one of most common causative factors
Mercury - MCS
Ciguatoxin (a type of poison found in reef fishes whose flesh is exposed to toxins produced by dinoflagellates (a type of plankton)) - CFS, ME
Heavy metal toxicity is most likely responsible for excessive immune system iNOS induction. Heavy metal toxicity, as well as impairing a number of processes in the body, also instigates an enormous increase in free radical formation. This oxidative stress on the body is a problem in of itself but it also triggers an inflammatory response from the body, partly in the form of iNOS induction, producing more Nitric Oxide.
Please note that heavy metal toxicity as a trigger does not just apply to cumulative toxicity through exposure, but can also apply to excessive or inappropriate chelation therapy for the removal of heavy metals. Excessively aggressive chelation, especially the use of mobilising agents, may increase levels of circulating heavy metals in the bloodstream, triggering more free radical production. Some symptoms of excessive detoxification including liver spots, which are swellings or boils (i.e. the body's inflammatory immune response to excessive liberated toxins). This may spiral out of control if the level of inflammation is already elevated or if sufficient breaks are not taken. If you are in the middle of a chelation programme and are suffering worse symptoms of inflammation, it is probably wise to take a break and focus more on antioxidant therapy until symptoms subside, as otherwise you may make it worse.
Of course, individual cases may well have more than one stressor and not necessarily as classified above. These comorbid conditions are fluid and vary in their progression and exact nature according to the individual.
According to Pall, excessive psychological stress over one's childhood or recent adulthood, or a single psycholgically traumatic incident is thought to significantly raise NO production and levels in the body. This is noted as the key driver in most Post Traumatic Stress Disorder (PTSD) cases and may play some role in CFS, ME and Fibromyalgia patients.
Inducible NO synthase (iNOS) is upregulated in lungs and liver during shock and plays a role for the generation of large amounts of NO during shock or following stimulation of tissues with a variety of proinflammatory mediators.
According to Pall, physical trauma is sometimes implicated in the onset of PTSD, CFS, ME and Fibromyalgia cases. Head and neck trauma is particularly relevant in Fibromyalgia cases, and head trauma oftenrelevant in PTDS cases.
Inducible NO synthase (iNOS) is upregulated in lungs and liver during shock and plays a role for the generation of large amounts of NO during shock or following stimulation of tissues with a variety of proinflammatory mediators.
According to Pall, ionising radiation exposure is implicated in the onset of CFS and ME in some patients, perhaps a cumulative effect of repeated exposures. Ionising radiation may come from a variety of sources, other than just medical or dental.
Pre-Existing Autoimmune Disease or Inflammatory Condition:
Whilst this is probably a chicken and egg situation, it is worth mentioning. Any type of inflammatory or autoimmune disease or condition will be characterised by increased levels of free radicals, including Nitric Oxide and Peroxynitrite. Such a condition in many cases may result in other parallel conditions arising, that are also fed by the Nitric Oxide/Peroxynitrite pathway. This is why such conditions are often referred to as being comorbid. CFS may arise from MCS, Fibromyalgia may arise from CFS or the other way around, over time. CFS may arise from Multiple Sclerosis or IBS. Pall notes that this is particularly relevant for Fibromyalgia, which may sometimes arise out of a pre-existing autoimmune disease.
Genetic Predisposition to Elevated Nitric Oxide Production:
Two genes have been identified in 5 studies as being implicated in CFS development, implicated in increased Nitric Oxide production. These are:
Defective CBG proteins have been associated with CFS cases, whereby the defective function of the CBG protein affects its ability to transport cortisol, predisposing such individuals to potentially developing CFS, along with many other possible acquired factors. Cortisol has a key role in lowering the induction of iNOS (immune system NO production enzyme), and lowered cortisol levels may be associated with elevated NO levels.
Angiotensin converting enzyme (ACE) gene
A polymorphism of this ACE gene has been associated with CFS and GWS in studies. Angiotensin II, the protein produced by the ACE protein, acts to elevate Superoxide levels.
In addition, another study from Rowe's Laboratory links orthostatic intolerance and CFS to Ehlers-Danlos Syndrome:
Ehlers-Danlos syndrome is a collection of diseases characterised by mutation in a subunit of the protein collagen or an enzyme that modifies the structure of collagen. Collagen is main structural protein in the body. Ehlers-Danlos mutations can affect vasculature, resulting in less effective perfusion (blood supply of O2 and nutrients into the tissues) of the elevated tissues of the body and resulting tissue hypoxia in these elevated tissues. Hypoxia can lead to Peroxynitrite production (i.e. through increased NO and O2- production). This may explain why some CFS patients prefer to lie down and remain in bed. However, it can work both ways, as blood perfusion and diffusion of O2 and CO2 may be impaired in the supine posture and may be improved in an upright posture, depending on brainstem function.
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).
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.
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:
Electron transport chain inside mitochondria: O2- is one of the intermediate byproducts of respiration and energy production. O2- levels may become elevated by the presence of Peroxynitrite (see below). Superoxide formation as an intermediate free radical during respiration is discussed on the Oxidative Stress page.
Uncoupled nitric oxide synthases (NOS): production of O2- by NOS occurs when levels of L-Arginine and/or BH4 are low and it is not possible to synthesise NO in sufficient quantities (as described above). Peroxynitrite oxidation of BH4 may be partly responsible for this increase in O2- production in place of NO production.
Xanthine Oxidase activity- Hypoxia (low O2 stress) increases the conversion of the Xanthine dehydrogenase enzyme to Xanthine Oxidase because of the effect of hypoxia on intracellular Ca2+ levels and energy metabolism. Xanthine oxidase generates O2- when it oxidises compounds such as hypoxanthine. Xanthine oxidase acivity may also be increased when cells are depleted of reduced GSH (Glutathione) (e.g. when cells are under oxidative stress). This may result in yet further O2- production. An increase in Xanthine Oxidase levels may thus result in increased O2- production. Hypoxia can also result in increased NO production from capillary budding - please see the NO section above. These two factors may result in elevated Peroxynitrite formation.
NADPH oxidase enzyme activity in Phagocytes (White Blood Cells) - Phagocytes consume O2 and use it to generate O2-, which is used by the NADPH oxidaze enzyme to generate a variety of different oxidants that are used to kill the fungi and bacteria that they ingest. This can be a substantial source of O2- and other oxidants in infected tissues. An increase in O2- production may thus result from the immune system's inflammatory response.
Cytochrome P450 enzymes - the decoupling reaction of the P450 catalytic cycle (addition of O2 to the Iron in the Heme group of the P450) results in the release of an O2- radical. However it is not thought that this is a significant factor in the NO/Peroxynitrite cycle.
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.
Genetic Predisposition to Elevated Superoxide Production:
Serotonin transporter gene
Certain forms of the Serotonin transporter gene may result in increased transport and consequently lower extracellular Serotonin levels, and are associated with CFS cases. This may lead to decreased HPA axis function, which in turn, may lead to lowered Cortisol secretion.
As discussed above in the section above on Genetic Predisposition to NO Production, a study from Rowe's Laboratory links orthostatic intolerance and CFS to Ehlers-Danlos Syndrome. Ehlers-Danlos syndrome is a collection of diseases characterised by mutation in a subunit of the protein collagen or an enzyme that modifies the structure of collagen. Collagen is main structural protein in the body. Ehlers-Danlos mutations can affect vasculature, resulting in less effective perfusion (blood supply of O2 and nutrients into the tissues) of the elevated tissues of the body and resulting tissue hypoxia in these elevated tissues. Hypoxia can lead to Peroxynitrite production (i.e. through increased NO and O2- production). This may explain why some CFS patients prefer to lie down and remain in bed. However, it can work both ways, as blood perfusion and diffusion of O2 and CO2 may be impaired in the supine posture and may be improved in an upright posture, depending on brainstem function.
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.
Mitochondrion - intracellular - inside the mitochondrial membrane, to protect the inner mitochondrial membrane from free radical damage from O2- produced during energy production and respiration.
Cytoplasm -intracellular - protects the inside of the cell (but outside of the mitochondria) from Superoxide damage
Secretory - extracellular - this type of SOD is secreted on the outside of cells and serves to neutralise SOD in extracellular spaces in the body.
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.
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).
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.
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.
ONOO- readily reacts nucleophilically with CO2, donating both of its bonded or shared electrons to form Nitrosoperoxycarbonate (ONOOCO2-). CO2 is of course produced by respiration as discussed on the Tissue Oxygenation and CFS page. CO2 is mainly in the form of Bicarbonate (HCO3-) in the blood and tissues, with a small amount dissolved as CO2 and also bonded to Hemoglobin.
ONOO- + CO2 -> ONOOCO2
Most of the ONOO- formed in the body from NO and O2-, does not remain as Peroxynitrite, but immediately reacts with the ubiquitous CO2 in the body, which is the predominant pathway for ONOO-. In other words, most of the free radical damage caused by Peroxynitrite is actually caused by the downstream products of Peroxynitrite, chiefly those formed from Nitrosperoxycarbonate (i.e. the Carbonate Radical CO3- discussed below), rather than the actual direction oxidation by ONOO- itself on the body's lipids and protein structures. However, for simplicity's sake, most commentators simply refer to 'Peroxynitrite' when they mean the actual downstream reacted products of Peroxynitrite.
Nitrosperoxycarbonate (ONOOCO2-) homolyzes (dissassociates) to form a Carbonate free radical (CO3-) and Nitrogen Dioxide (NO2).
onooco2- -> CO3- + NO2
CO3- is the radical form of Carbonate which has the chemical formula CO3-- or CO3(2-). In other words the Carbonate Radical has on less electron (i.e. it has one unpaired outer electron), making it hugely more reactive and a free radical. Both the Carbonate Radical and Nitrogen Dioxide Radical are responsible for the majority of the Peroxynitrite-related oxidative stress and damage in the body. The Carbonate Radical however could be considered the more reactive and nastier of the two.
According to Pall, Peroxynitrous acid, the acidic form of Peroxynitrite (HOONO) is present in the cell in approximately 1:3 of Peroxynitrous acid to Peroxynitrite (which has not reacted with CO2). It is formed when the Peroxynitrite ONOO- anion is in the presence of an acid (i.e. the H+ cation), the two bonding to form Peroxynitrous acid. It is relatively unstable. It can be written (expressed) as HOONO or ONOOH.
Perxoynitrous acid (HOONO) breaks down or homolyzes to form caged radical Nitrogen Dioxide (NO2) and the Hydroxyl (Free) Radical (OH). Both of these radicals can react to cause damage to important molecular structures in the body.
ONOO- + H+ -> HOONO -> NO2. + OH.
Approximately 2/3rds of the NO2 and OH radicals produced from HOONO exchange an electron (i.e. electron transfer) to produce the unstable NItronium cation (NO2+) and Hydroxide anion (OH-).
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.
Inactivation of Iron-Sulphur Proteins:
ONOO- and its related products (including NO) can inactivate iron-sulphur proteins. The most important of which arguably are those found in certain Mitochondrial enzymes that are part of the Krebs Cycle (Citric Acid Cycle), e.g. Aconitase enzyme. The damage to these enzymes (proteins) is irreversible and the only way their functionality can be restored is through resynthesis of these proteins. Damage to these proteins may result in a bottleneck in the Krebs Cycle and accumulations of both cis-aconitate and its precursor citrate. Succinate dehydrogenase (a.k.a. Succinate-coenyme Q reductase (SQR) or Complex II) is another example, and it participates in both the citric acid cycle and the electron transport chain.
Several types of DNA damage can be inflicted including the nicking of of the backbone of DNA chains. These nicks stimulate the poly (ADP-ribose) polymerase enzyem, which uses Active Vitamin B3 (NAD) as a substrate. NADH is the reduced form of Active B3 that is involved in the electron transport chain in mitochondria. Therefore elevatd poly (ADP-ribose) polymerase enzyme production on account of the DNA damage caused by ONOO- can lead to a depletion of the pools of NADH/NAD that are normally used in mitochondrial function, thus heavily impacting ATP availability and energy levels. NADH supplementation may therefore be beneficial to overcome this deficit in the mitochondria. NADH does stimulate NO production, but if its levels are very low in the mitochondria, then is not likely an issue until levels greatly exceed requirements (rather difficult but possible).
Oxidative Chains Reactions, Including Lipid Peroxidation:
ONOO- and its products can instigate lipid peroxidation, i.e. oxidation of the lipids in biological and cellular membranes, in particular, mitochondrial membranes. Cell membranes are made up Essential Fatty Acids and Phosphatidyl Choline on the whole, both of which are polyunsaturated, and can be easily oxidised. Lipid peroxidation causes many changes in cellular functioning, and lipid peroxidation of the mitochondrial membranes can affect mitochondrial functioning. The latter is more likely to be caused by Superoxide than ONOO-, although ONOO- can still be significant.
The Superoxide Dismutase (SOD) protective enzyme as described above is a protective antioxidant enzyme used to neutralise Superoxide. 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.
Nitration and Oxidation of Proteins:
Modification of proteins by oxidation and nitration to form products such as Protein Carbonyls and 3-Nitrotyrosines
Stimulation of NF-kB DNA transcription factor:
The oxidant products of ONOO-, as well as other oxidants and free radicals, stimulate the activity of Transcription Factor NF-kB and also AP-1. NF-b is involved in cellular responses to stimuli such as stress, cytokines, free radicals, UV irradiation, oxidised LDL and bacterial or viral antigens. AP-1 is also involved in cellular responses to many of the above. According to Pall, Reduced Glutathione and its precursors (N-Acetyl-Cysteine), Lipoic Acid, Vitamin C, alpha-tocopherol and numerous phenolic antioxidants (inc. flavonoids) and Selenium all act to lower NF-kB activity.
Oxidation of Hemoglobin:
According go Susanna Herold and Kalinga Shivashankar's article 'Metmyoglobin and Methemoglobin Catalyze the Isomerization of Peroxynitrite to Nitrate' Biochemistry, 2003, 42 (47), pp 14036-14046:
'Hemoproteins, in particular, myoglobin and hemoglobin, are among the major targets of peroxynitrite in vivo. The oxygenated forms of these proteins are oxidized by peroxynitrite to their corresponding iron(iii) forms (metMb and metHb)...inally, we showed that different forms of Mb and Hb protect free tyrosine from peroxynitrite-mediated nitration'
ONOO- and its products are implicated in the damage of the blood-brain barrier. Lipoic acid helps to restore the blood-brain barrier function.
Early Apoptosis (Programmed Cell Death):
Elevated ONOO- can result in early programmed cell death, known as apoptosis. Apoptosis is a sequential and ordered process, cells going through a certain sequence of events that leads to their eventual death. Apoptosis serves a number of functions, namely cell termination (of cells that are damaged beyond repair, virally infected etc. or undergoing stressful conditions such as starvation), homeostasis (balancing cell division with cell death in each individual cell) and tissue development. It serves additional roles in early tissue development in minors and the development of the immune system (to avoid auto-immune diseases and to eliminate precancerous cells) etc. Apoptosis is also involved in the death of cells in neurodegenerative diseases.
Necrosis on the other hand is unprogrammed cell death which involves an inflammatory reaction to the cell death, and whereby the cell has not gone through its normal sequence. Think of Necrosis as the cellular equivalent of being murdered, as opposed to Apoptosis as dieing of old age.
The NO/ONOO- cycle (i.e. elevated ONOO-, NO and O2- levels and excessive NMDA activity) play a role in cell death in certain neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntingdon's disease. Peroxynitrite is known to induce apoptosis in a large number of cell types, and as a result, can be expected to increase apoptotic cell death rates in these multisystem illnesses. Apoptosis is noted in a number of other multi-system illnesses as is believed to be due to elevated levels of 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.
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.
Pall had made the following suggestions to avoid exacerbating the NO/ONOO- cycle. These include the avoidance of:
Exposure to chemicals that produce sensitivity responses, especially in Multiple Chemical Sensitivity patients
Excessive exercise and the associated post-exertional malaise as this upregulates the cycle biochemistry, especially those with CFS symptoms, but also relevant for ME and Fibromyalgia patients who do not feel tired round the clock but have post-exertional malaise.
Foods that produce an immune system response (whether an allergy or intolerance). Exposure to such antigens increases NO levels.
Excitoxins, especially Glutamate and aspartate (discussed on the Food Allergy page) as these stimulate the NMDA receptors.
Psychological stress, especially for Post Traumatic Stress Disorder (PTSD) sufferers.
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:
Avoid raising one's L-Arginine levels above normal, as this may result in increased NO production - this might include either L-Arginine supplements or consuming Arginine-rich foods. However, if L-Arginine levels are low, then one may benefit from L-Arginine supplementation boosting its role in neurotransmitter production in general. An Amino Acid Profile would confirm one's present L-Arginine level.
Exposure to strong electromagnetic fields.
Exposure to elevated levels of heavy metals - chelating therapy can help reduce the levels of circulating heavy metals if done conservatively. Chelation therapy can however aggravate inflammation. If you have recently been undergoing intensive chelation therapy, especially with mobilising agents, take a break for a while, and reconsider the pace, number of breaks and dosages of your chelation therapy when you recommence. A congested bowel or congested skin may also keep inflammation levels high on account of the retained toxins, and keeping the bowel moving, cleansing the bowel of toxins with absorbants, and also cleansing the skin with clays etc. may also help reduce inflammation slightly; to avoid the build of heavy metals in the organs of elimination.
Electromagnetic treatment devices, including magnetic, Teslar or Far Infrared (FIR), if one's inflammation is being fuelled by high circulating levels of heavy metals, as these may increase the amount of mobilisation of heavy metals. However, if one does not have significantly above average levels of circulating heavy metals, then such treatments or devices may actually help!
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.
Lipoic Acid - may deplete biotin levels, so supplementation with biotin is preferable. Most ALA supplements contain added biotin. Powerful antioxidant (regenerates Glutathione) and a chelating agent (crosses the blood brain barrier). Lipid soluble. Also directly scavenge free radicals and oxidants, including ONOO-, singlet Oxygen, O2-, Peroxyl radicals and breakdown products of ONOO-. Useful in the treatment of diabetes.
Betaine (TriMethylGlycine or TMG) - lowers reductive stress (if applicable).
Coenzyme Q10 - antioxidant (helping to scavenge ONOO- breakdown products) and mitochondrial cofactor (electron transport chain - mitochondrial support). Best taken in morning or lunchtime.
Hydroxocobalamin (Active Vitamin B12 Precursor) - nebulised, inhaled (nasal spray) or sublingual (dissolve under tongue). Nasal spray is reputed to be an effective method of delivery in lieu of an Intramuscular (IM) injection. Useful in methylation (Glutathione production). Nitric Oxide scavenger.
Folic Acid (B9) - useful to support methylation (Glutathione production). Folate is known to increase uncoupling of NOS activity, thus lowering production of NOS-dependent Superoxide. Active Folate (5-MTHF) helps to regenerate BH4. It also binds to eNOS in place of BH4, decreasing the enzyme uncoupling.
Pyridoxal-5-Phosphate (P5P or Active B6) - involved in GABA production, helping to reduce NMDA activity. B6 is often critically low in CFS patients. Performs many other roles in energy production and the endocrine system.
Riboflavin 5'-Phosphate (FMN or Active B2) - supports energy production. One of the cofactors that forms Glutathione reductase contains FAD (bioactive B2). FMN is the precursor to FAD. Important in reduction of oxidised glutathione back to reduced form.
Gamma-Tocopherol (Vitamin E) - a source of mixed, natural Tocopherols is preferred (including a high Gamma-tocopherol content), not synthetic Tocopherols. Gamma-Tocophernol is an Antioxidant and Peroxynitrite scavenger. Natural Vitamin also contains Tocotrienols. Tocopherols, particular, Gamma form, help to lower excitotoxicity and thus NMDA activity. High alpha-Tocopherol content, e.g. in synthetic sources, tends to lower bodily Gamma-Tocopherol levels and thus lower peroxynitrite scavenging abilities of the body. Gamma-Tocopherol supplements are often known as 'Gamma E'
Ascorbic Acid (Vitamin C) - buffered - e.g. 250mg/day doses. Helps to regenerate BH4 and may help to lower NOS uncoupling.
Carotenoids - including lycopene, lutein and Beta Carotene (Vitamin A Precursor). Lipid soluble antioxidants - Peroxynitrite scavengers. Lycopene is considered a superior ONOO- scavenger than Beta Carotene. High dose Beta Carotene may have adverse effects. Spirulina is rich in Beta Carotene.
Flavanoids - plant phenolic antioxidants - from 4 different sources: Ginkgo biloba extract, Cranberry extract, Silymarin (Milk Thistle) and Bilberry Extract. Flavonoids are known to lower NF-kB activity and act as potent chain-breaking antioxidants and are effective at regenerating other potent antioxidants. Water and lipid soluble, so can act as antioxidants in water and lipid compartments in the body. One class, anthocyanins and proanthocyanidins are red, purple or blue in colour, and found in purple coloured berries and vegetables and are potent superoxide scavengers. Many other flavonoids have a pale yellow colour. Green tea (extract's) flavonoids have been shown to scavenge peroxynitrite, superoxide and nitric oxide. Soy, hawthorn and olive extract have been shown to scavenge superoxide. Silymarin (Milk Thistle extract) is reported to scavenge peroxynitrite and also increase synthesis of SOD. Flavonoids are absorbed rapidly from food but also excreted rapidly by kidneys and levels may peak 2 hours after eating. It is therefore optimal to consume smaller amounts of flavonoid sources throughout the day or several times a day. [my comment - there are many sources of some of these antioxidants. Oligomeric ProanthoCyanidins (OPCs) are a class of polyphenol antioxidant that have exceptional free radical scavenging abilities and help to lower blood pressure. They are found in high concentrations in Grape Seed Extract. OPCs help to prevent iNOS from activating and from knocking out the tetrahydrobiopterin step of eNOS. Resveratrol is a another type of polyphenol, found in (red) Grape skins. It is a powerful antioxidant and another compound used to lower blood pressure. It works by upregulating SIRT1 gene activity which in turn downregulates NF-kB and inflammation (thus allowing mitochondria to repair), which both help prevent iNOS from activating.
Magnesium Malate - Mg lowers NMDA activity (by counteracting the ingress of Calcium ions) and is involved in ATP activation and enzyme activation. Usually chronically deficient in CFS, ME and FMS patients. Malate (Malic Acid) is a Krebs cycle cofactor for energy production (found in apples), often low in CFS patients.
Zinc - modest dose - component of SOD. Also used in immune system metalloproteins. Helps to inhibit NO release from NMDA stimulation by making NMDA receptor depolarisation more difficult. Antioxidant. Displaced by heavy metals.
Manganese - low dose - component of SOD
Copper - low dose - component of SOD
Selenium - as Selenium-grown yeast (i.e. Selenomethionine)- used in antioxidant metalloproteins (e.g. peroxidase enzymes) - good ONOO- scavengers. Se is often low in CFS and MCS patients.
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.
5-MTHF (Active B9) in place of Folic Acid - Folic acid is the synthetic form and has to be converted via a number of steps into 5-MTHF which is the form actually used by the body. Folic acid in itself serves no biological function and is merely a precursor. These steps require ATP and effective enymatic steps, and some people are genetically predisposed not to be able to convert Folic acid/Folate effectively.
Methyl-B12 in place of Hydroxo-B12? Methyl-B12 is the active end form of B12 that is used in methylation and the production of Glutathione. Hydroxo-B12 is a precursor and must be converted into Methyl-B12 before it can be used. This requires ATP. Pall is unclear in his book whether Hydroxocobalamin (Hydroxo-B12) is recommended as it is a precursor to both Methyl-B12 (used in methylation) and Adenosyl-B12 (used in energy production) and thus is convenient to take rather than supplementing both of these forms of B12, or whether it is because Hydroxo-B12 is more effective as an NO scavenger than any of the actual end forms of active B12 that are used in the body. The 2009 study by Weinberg et al., 'Inhibition of Nitric Oxide Synthase by Cobalamins and Cobinamides' suggests that whilst Hydroxo-B12 is much more effective than Methyl-B12 or Adenosyl-B12 in inhibiting NOS:
'Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity...Hydroxocobalamin (OH-Cbl...potently inhibited all isoforms [of NOS], whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect.'
Thus whilst Methyl-B12 may be more effective in boosting Glutathione production directly, Hydroxo-B12 may be the preferred form in this instance on account of its superior NOS inhibiting qualities.
Betaine HCl in place of Betaine - whilst Betaine can be useful, it makes sense to take it in HCl (acidic) format and use it as a supplement to take with meals to raise stomach acidity levels to aid digestion. The majority of CFS patients do not produce enough stomach acid. Betaine HCl lowers reductive stress (lack of Oxygen to oxidise certain molecules). If you produce sufficient stomach acid, then take the Betaine form instead.
NAD / NADH (Active B3) - we have seen above how NADH levels can become depleted in individuals with raised Peroxynitrite levels and supplementation is useful to support mitochondrial function.
Cocarboxylase (TDP orTTP) (Active B1) - useful for supporting mitochondrial function and energy production.
Sulphoraphane Glucosinolate (SGS - from Broccoli seeds/sprouts) and Ecklavia cava (brown algae) extract (a.k.a. ECE, SEANOL-F or FibroBoost). These are probably two of the most powerful and long lasting naturally occurring antioxidants there are. Daily supplementation with one or both of these would offer round the clock protection against oxidant and free radical damage from any of the ROS mentioned above. Please see the Nutritional page for more information.
Astaxanthin - a type of carotenoid found in Haematococcus Pluvialis microalgae. It is a potent antioxidant. It is known to be the most powerful naturally occurring antioxidant, having 550 times the antioxidant activity of Vitamin E and 10 times the activity of Beta Carotene.
DMAE - a precursor to Choline and the neurotransmitter Acetylcholine - is also a powerful OH. free radical scavenger. Can also be taken in the drug form Centrophenoxine.
Oxidative Stress Relief (OSR) is a new synthetic chelating agent which extremely powerful antioxidant properties that may well help to reduce ONOO- based inflammation as well as remove any Mercury that might be contributing to the inflammation.
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.
Food as Therapy. Try to eat antioxidant-rich food as possible (high ORAC values). Avoid any foods associated with allergic or intolerance reactions as they will increase inflammation which is the opposite of what we want. Rapidly growing foods (e.g. sprouts) or storage compartments (e.g. seeds) are highly nutritious. Correct balance of Omega 3 and 6 EFAs.
Omega 3 Essential Fatty Acids - DHA and EPA from fish. Oily fish may contain elevated Mercury levels. If you buy a supplement, ensure it is screened for heavy metal levels. Poweful antioxidants on account of large number of unsaturated bonds. Useful in brainfunction and lowering iNOS induction by lowering NF-kB activity.
Phospholipids and Glycolipids - for repairing oxidative damage to the inner mitochondrial membranes. Useful in lowering reductive stress.
Acetyl L-Carnitine (ALC) - better absorbed than L-Carnitine. A powerful antioxidant and a mitochondrial cofactor (transporter of ATP). It helps to lower the mitochondrial phase transition (which leads to apoptotic cell death), thus playing a protective role in mitochondria. It also helps to transport fatty acids into the mitochondria. The inner mitochondrial membranes' cardiolipin are made up predominantly of fatty acids. (90+% Omega 6 fatty acids).
L-Taurine - antioxidant amino acid. Neurotransmitter (inhibitory) - helps lower NMDA activity. Lowers intracellular Calcium levels (thus reducing NO production). Also involved in transport of minerals into cells, e.g. Magnesium. Often wasted in the urine to save depletion of Magnesium levels. Critically important to supplement in many CFS cases.
L-Carnosine - antioxidant amino acid. Antiglycination agent - reverses some of the damage produced by excessive sugar to proteins. Concentrates in brain and muscles (particularly the heart), performing a protective function there against oxidants. An ONOO- scavenger.
Creatine - stores energy in brain and muscle tissues, in form of Creatine (Mono)Phosphate. It appears to stimulate transport of Glutamate into Glial cells, thus lowering excitotoxicity produced by Glutamate (and thus lowering NMDA activity).
Cat's Claw - antiviral herb that helps also to lower NF-kB activity. Feverfew may also help (contains melatonin).
Curcumin - yellow pigment in Tumeric. A polyphenolic (chain breaking) antioxidant, aand lowers NF-kB activity. Stimulates the synthesis of Glutathione. A useful peroxynitrite scavenger and also complexes with Copper or Manganese to form an SOD-mimic molecule (metabolises Superoxide).
Panax ginseng extracts - ginsenosides have been reported to lower Vanilloid receptor activity. Adaptogenic herb that stimulates the adrenal glands.
Chlorella (green algae) and blue-green algae (e.g. Spirulina). Useful for detoxification, nutrition and as peroxynitrite scavengers on account of their rich carotenoid and vitamin C antioxidant content. Blue-green algaes contain phycocyanins are potent peroxynitrite scavengers. Both however do contain Glutamic acid and Aspartic acid, Spirulina containing about twice the amount compared with Chlorella. Use in moderation.
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.
Inosine - Uric Acid is antioxidant compound in human blood, created when the body breaks down purine nucleotides, a peroxynitrite scavenger and prevents ONOO- mediated breakdown of blood-brain barrier; and which lowers uncoupling of NOS that is produced by ONOO- mediated oxidation of BH4. Uric acid levels are often low in inflammatory diseases. Gout results in elevated uric acid levels. Uric acid cannot be taken orally as it is broken down in the GI tract but the purine-containing Inosine can be. Inosine is a purine nucleoside and constituent of nucleic acids. High purine foods can cause kidney stones. Inosine is converted to uric acid by the xanthine dehydrogenase/xanthine oxidase enzymes. The latter enzyme also creates O2- in the process, so Inosine's role is not completely clear. It may perhaps be worth considering a supplement that contains Biopterin, as boosting purine levels is one mechanism for boosting BH4 levels, e.g. Norival by Cardiovascular Research.
Porphyrins - act as SOD mimics. Experimental only! Involved in Heme synthesis. Elevated levels can occur on account of heavy metal toxicity (interfering with Hemoglobin production). Porphyria is a hereditary condition of elevated porphyrin levels to the point of chronic toxicity and even death.
Hyperbaric Oxygen Therapy - increased oxygen competes with NO and lowers the inhibition of NO of cytochrome oxidase enzymes used in energy production. May allow tissues to respire and produce CO2 at a higher rate, producing more energy - and requiring more mitochondrial cofactors. May exacerbate oxidative stress on account of additional tissue Hydrogen Peroxide production. This may increase BH4 production and may decrease uncoupling of NOS. Must take antioxidants at the same time to guard against oxidative damage. Please see Cheney's observations on CFS patients who PFOs on the Cardiac Insufficiency page and the Tissue Oxygenation page.
Hydrogen Peroxide therapy - increases BH4 levels - but the downside is the additional oxidative stress it puts on the body.
Minocycline - a type of tetracycline antibiotic with antioxidant and anti-inflammatory properties, including scavenging of ONOO- or its breakdown products. Crosses blood-brain barrier.
Ebselen - a synthetic organic Selenium compound currently under drug testing. A potent ONOO- and free radical scavenger. Also acts as a Glutathione peroxidase mimic, substituting for Glutathione Peroxidase in eliminating organic peroxides. Regenerates other antioxidants.
Riluzole - drugs that is a Sodium channel blocker and indirectly lowers Glutamate release (thus lowering NMDA activity).
Guaifenesin - derived from a tree bark extract called Guaiacum. Drug that helps to lower Vanilloid receptor activity as well as as an expectorant (helps loosen and liquefy mucus). Used in treatment of Fibromyalgia (with variable success).
NMDA Antagonist drugs - dextromethorphan, memantine, ketamine and flupirtine.
Paroxetine (SSRI) - used for treatment of MCS and PTSD, which is also known to lower NO synthesis.
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.
Methods of reducing elevated Peroxynitrite levels:
Dr Paul Cheney (in 2004) has suggested a number of means of fighting high Peroxynitrite levels.
Stimulating GABA production - which in turn downregulates the NMDA receptor, reducing Nitric Oxide production. Herbs and amino acids that can stimulate GABA production and the endocrine system in general are discussed on the Endocrine page. Cheney does not actually discuss natural means of stabilising neurotransmitter production in his article.
Increasing bodily CO2 levels - by rebreathing or by going below sea level (or presumably breathing a higher partial pressure of O2 - causing one to breathe less and thus build up more CO2); CO2 being a scavenger of Peroxynitrite. CO2 reacts with Peroxynitrite (ONOO-) to form Nitrosoperoxycarbonate (ONOOCO2-). Conversely, being in a plane with a reduced pressure compared to ground pressure of air is reported to make CFS patients feel worse. Cheney in this interview assumes it is the CO2 that is making the difference here, rather than the oxygen levels, which he does not discuss. Cheney also argues elsewhere that as the body's metabolic rate has slowed down, the body requires less O2.
Whilst CO2 may be a good scavenger of Peroxynitrite, it is also toxic and contributes to Acidosis in the body. It should be noted that deep breathing will also probably stimulate GABA and Dopamine production, to downregulate various systems including Nitric Oxide production (as described above). This is discussed in relation to metabolism compensation in the Comments section towards the end of this page.
However, in my opinion, it is much more likely that a higher partial pressure of oxygen (i.e. increased air pressure or breathing pure O2) is actually making the patients feel better as their tissues are actually deficient in oxygen, rather than needing more CO2 to fight peroxynitrite levels (or lower pH levels). I have understood from various sources that CFS patients actually have too little O2 and too much CO2 in their blood (i.e. too low a pH rather than too high) and would probably recommend the complete opposite of Cheney in this respect, i.e. deep breathing and avoiding rebreathing like the plague. This is discussed further in the final section on this page.
I have myself gained considerable benefit from deep breathing exercises using either air or pure oxygen during various stages of my CFS, and this was not breathed in a shallow manner, but deeply, in order to flush out as much CO2 as possible and to oxygenate the body as much as possible. However, perhaps, as Cheney states, above, I was at this stage within the threshold of oxygenation, where additional oxygen was still being beneficial rather than detrimental. It may not be the same for all CFS patients, but I am not totally convinced.
Increasing Uric Acid levels. Uric acid is a powerful scavenger of peroxynitrite. Cheney has found uric acid levels in the CFS patients he has examined to be extremely low. Uric acid is formed from RNA and DNA metabolism within the body - through apoptosis (the programmed death of cells and their consumption by the body) or by fasting (by consuming the body's own cells). In terms of external sources of RNA, some excellent sources include Spirulina algae, uncontaminated raw meats or fish (such as Sushi) - cooking destroying RNA and DNA, raw milk, non-denatured whey protein, eggs, fresh/young foods (nuts, seeds, sprouts, baby lettuce etc.). Clearly many sources of raw meat and fish are contaminated and may contain elevated levels of parasites. However, one should also consider that many raw vegetables also contain parasites which can be problematic if they are not washed properly. Excessive raw vegetable intake may also be problematic from a 'cold energy' perspective. If vegetables are to be cooked steaming is preferable, but microwaving should be avoided (for a variety of reasons!) as it damages the RNA more than any other cooking method. Please see the Digestive Disorders page for information on Spirulina and also non-denatured whey protein drinks.
Consume Reduced HDL (good) Cholesterol. When HDL cholesterol binds with Peroxynitrite, it produces oxidised LDL ('bad' cholesterol). Please see the Nutritional page for more information on fatty acids. Heating or cooking HDL however oxidises it, so we are interested in the unoxidised or heat treated form. Good sources of reduced HDL include untreated dairy products (e.g. raw milk) or non-denatured whey protein.
Methods of reducing elevated Peroxynitrite and Superoxide levels:
Cheney (in 2004) has suggested a number of means of fighting high Peroxynitrite and Superoxide levels.
Assisting Glutathione Peroxidase activation by taking Zinc and Selenium supplements.
Taking Coenzyme Q-10 and/or Idebenone. Idebenone is an organic compound of the quinone family and is advertised as a synthetic equivalent to Coenzyme Q10. It is believed to have similar properties to Coenzyme Q10, but perhaps Q10 is preferable. Please see the Mitochondrial page for more information.
In Dr Cheney's Fairfax, Virginia seminar from 25 April 2009, he states the taking Coenzyme Q10 is actually 'toxic' to CFS patients and can harm the heart (and the increase oxidative stress on the heart). This is a 360 degree turnaround in terms of recommendation and counter intuitive to me.
taking other antioxidants, in particular Proanthocyanidins or bioflavinoids, in particular grape skins or pycnogenol. Please see the Nutritional page for more information. There are many other powerful antioxidants that Cheney does not mention including GliSODin, which helps to stimulate SOD production. Although Cheney mentions Lipoic Acid as a protective mechanism against Superoxide leaking out of the mitochondria, he does not include supplemental Lipoic Acid as a useful antioxidant to take.
Methods of blocking Nitric Oxide Production:
Cheney (in 2004) has suggested a number of means of blocking Nitric Oxide production.
Hemoglobin - the iron-containing oxygen transport metalloprotein found in red blood cells. Hemoglobin is the best endogenous oxygen scavenger of nitric oxide. When hemoglobin combines with nitric oxide, it becomes bent, causing the red blood cells to deform. This can be seen in a live blood microscopy. Erythrocyte Sedimentation Rate (ESR) haematology tests also tend to show a low sedimentation rate in CFS patients, suggesting a high degree of inflammation/red blood cell deformation and/or pain. The Laboratory Textbook of Medicine states that only two other diseases besides Sickle Cell Anemia (a genetic hemoglobinopathy), and they are CFS and Idiopathic Cardiomyopathy. In short, the extent of deformation of red blood cells is an indicator of inflammation due to excessive Nitric Oxide in the body, as well as potentially indicative of other sources of inflammation, for example, other sources of oxidative stress, dehydration and/or fatty acid imbalances etc.
During the course of CFS treatment, in my experience, a patient's red blood cells may go from a high degree of inflammation/deformation to virtually none whatsoever (for example, taking Magnesium supplements, antioxidants and/or Essential Fatty Acids), whilst still retaining many of the CFS symptoms. It is clearly one thing to say that high nitric oxide levels in the body were a major factor to explain the onset of mitochondrial function and its downstream effects, but it is quite another to simply address the nitric oxide issues with antioxidants and other means, and expect the condition to reverse itself, as all the downstream effects have to be addressed for the body as a whole to improve. One cannot simply remove the original cause and assume that full recovery will immediately follow without any other assistance. So it is perhaps not quite as straightforward as Cheney has implied.
Vitamin B12: This B-vitamin binds very strongly with Nitric Oxide. Cheney has recommended Hydroxycobalamin injections. In general, I recommend heavy Methyl-cobalamin for heavy supplementation as it is the form used in Homocysteine metabolism. B12 can also help to promote homocysteine metabolism and increase Glutathione production, so that this too can reduce the amount of superoxide that might leak from one's mitochondria. Cheney has not really delved deeply into promoting Glutathione production in this interview/paper.
Magnesium: Magnesium blocks the production of nitric oxide by calcium channel blockage. Magnesium is also an essential nutritional element that is frequently chronically low in CFS, M.E. and Fibromyalgia patients. Magnesium also helps to protect against excessive glutamate toxicity. Cheney recommends Magnesium Sulphate and Taurine injections. MgSO4 is the usualy form that Magnesium is injected as, but when taken orally, chelated Magnesium is preferable. As discussed above, Taurine is often wasted by CFS sufferers and increases the ability to absorb Magnesium. See the Afterload section on the Cardiac page for more information on applications for Magnesium supplementation.
In Cheney's 2006 seminar, he additionally recommends the following treatments, specifically relating to Peroxynitrite and Nitric Oxide management.
Glutathione. GSH's main role is as an antioxidant, protecting the body from oxidative damage (free radicals), but also to bind to heavy metals in the body, for subsequent removal in the liver (hopefully). It is also according to Cheney the most potent anti-viral compound in the body, and inhibits viral replication, being the most efficient method of combatting viruses. Please see the Liver Function page for more information. Glutathione is believed to generally assist in adrenal/steroid hormone production.
'Glutathione (GSH) is a tripeptide. It contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side chain. Glutathione, an antioxidant, protects cells from toxins such as free radicals. Thiol groups are kept in a reduced state at a concentration of approximately ~5 mM in animal cells. In effect, glutathione reduces any disulfide bond formed within cytoplasmic proteins to cysteines by acting as an electron donor. In the process, glutathione is converted to its oxidized form glutathione disulfide (GSSG). Glutathione is found almost exclusively in its reduced form, since the enzyme that reverts it from its oxidized form, glutathione reductase, is constitutively active and inducible upon oxidative stress. In fact, the ratio of reduced glutathione to oxidized glutathione within cells is often used scientifically as a measure of cellular toxicity.'
Cheney believes that direct Glutathione injections are very useful, but they should not be used too often (i.e. maximum of 3 times a week). He stated that he observed a decline in the patient's condition upon receiving a daily Glutathione injection. My own practitioner advised that one might feel slightly unwell after a Glutathione (and Phosphatidyl Choline) injection, on account on the immediate increase in detoxification function. But over a period of weeks or months, one would notice a huge improvement in detoxification of heavy metals and also wellbeing and energy levels. I only had such an injection once a week for a period of 6 months or so. T. Michael Culp has a similar view to Cheney, that Glutathione supplementation is very useful, but only in the right quantities required by the body. The body may not be able to handle the downstream effects of too much Glutathione, and only a small amount may be required. His preferred method of administering Glutathione is 100mg capsules of Tyler Recancostat, as opposed to injections. Injections may introduce large amounts suddenly into the body causing a kind of 'shock' to the body. Cheney's warning is probably not an issue for most people as it would be ludicrously expensive to have daily Glutathione injections!
According to Cheney, it is the reduced (i.e. unoxidised form) of Glutathione (GSH) that one wants to increase in the body, but because of a lack of energy in the body, Glutathione tends to end up as the oxidised form (GSSG). Too frequent Glutathione injections he states result in too much of the oxidised form of Glutathione building up in the body. He states that this results in the Metallothionein molecules 'opening up and releasing Heavy Metals'. He did not elaborate on the specifics in his seminar, which would have been helpful, but repeated a number of times these generalised comments. I do not however follow the logic here at all.
'[Metallothionein], serves a role as guardian preventing toxic metals from gaining entrance to our body and brain by binding to these metals at the surface of the GI tract and at the blood-brain barrier. This barrier protects the brain from dangerous substances that may be circulating in our blood stream.'
He similarly stated that taking Glutathione precursors such as NAC has a similar negative effect, and that two patients committed suicide after being recommended 1000mg of NAC per day. It is not clear from his seminar whether they were his patients or not, or indeed whether there were other predisposing factors. Indeed, there are many different forms of Cysteine that can be ingested, not just NAC, and it depends on what the body's actual Cysteine levels are, if there is a requirement for Cysteine or not (there usually is a deficit in CFS cases) and indeed whether the body might not prefer another form or even stabilised Glutathione itself (e.g. Tyler Recancostat). If Cysteine levels are already adequate, then taking additional Cysteine in the form of NAC might cause an amino acid imbalance, but that would probably not explain suicide. Everyone's body reacts differently. This is something that could have been determined using muscle testing however. I have myself taken more than this daily dosage of NAC and experienced no negative side effects whatsoever. Presumably, by Cheney's logic, the recommendation to avoid cysteine containing Glutathione precursors would also apply to non-denatured whey protein, MSM and Lipoic Acid which are also Glutathione precursors or assist in Glutathione production. Cheney does however recommend non-denatured whey protein as one protocol for increased DNA and RNA intake (as discussed elsewhere on this page). So there seems to be some contradiction. Cheney has also recommended MSM, for example in his October 2001 seminar, outlined below.
In Dr Cheney's Fairfax, Virginia seminar from 25 April 2009, however, he seems to have evolved or changed his opinion, believing that the methylation cofactors, Methyl-B12 and 5-MTHF (discussed on the Nutritional Deficiencies page, are 'toxic' to CFS patients as it results in more oxidation and free radical damage. However, those patients with a methylation blockage are not able to produce Glutathione adequately. Glutathione is one of the two most important antioxidant enzymes in the body to prevent oxidation. So I cannot see the logic in the statement and is far from convinced.
In addition, a study by the University of Oxford - Department of Cardiovascular Medicine - has found that 5-MTHF 'rapidly improves endothelial function and decreases superoxide production in human vessels: effects on vascular tetrahydrobiopterin availability and endothelial nitric oxide synthase coupling...Futhermore, 5-MTHF was a strong peroxynitrite scavenger...' These findings in other words completely contradict Cheney's latest views that 5-MTHF supplementation will lead to more oxidative damage through more superoxide production.
Clearly, each case is different and needs to be considered on its own merits, and making this kind of generalised statement is not clever in my opinion.
Hawthorn Leaft Extract - an anti-inflammatory and antioxidant, containing Flavonoids. Also has a role in cardiac support. Discussed on the Cardiac Insufficiency page. As a mobilising agent for heavy metals, it may also potentially increase levels of inflammation rather than decrease them however.
Nexavir, formerly known as Kutapressin, is a prescription drug produced from pig's livers by Nexco Pharma. It is believed to be anti-inflammatory and anti-viral. In Cheney's application it is an enzyme activator, immuno-modulator and anti-viral drug for combatting specific viral infections. I cannot specifically comment on this drug, but in general does not support the use of anti-viral or immuno-modulating drugs unless as a last resort, and instead prefers the use of anti-viral herbs and oils. http://phoenix-cfs.org/TreatmentNexavirCFS.htm
Artesunate is an anti-malarial drug used to reduce egg production in Schistosoma haematobium infection. Artesunate is prepared from the malarial drug Dihydroartemisinin (DHA - not to be confused with docosahexaenoic acid, the Omega 3 EFA) by reacting it with succinic acid anhydride. Cheney first advocated the use of this anti-malarial drug in treating CFS patients in his 25 April 2009 Virginia seminar. He clais that it is a powerful redox inhibitor and shifts the body's redox state to normal, and is also useful as an antiviral treatment, active against all herpes viruses. How much of this success is down to its redox capabilities and how much is down to its antiviral properties, I am unable to comment. Artesunate is usually administered IV. http://en.wikipedia.org/wiki/Artesunate
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.
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.
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.
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.
NOS byproduct tests
NO and the amino acid Citruline are created by the action of NOS on the substrate L-Arginine and O2 in the presence of the B-vitamin (B3) cofactor (reducing agent) NADPH. One molecule of Citrulline is produced for every molecule of NO. Whilst NO has a half life of only one second and cannot be measure directly, it is possible to measure the amount of Citruline produced, as Citrulline is relatively stable.
Thus it is possible to differentiate between average or normal levels of Citrulline and elevated levels associated with elevated NO production - making Citrulline. Whether the additional NO produced has reacted directly as a free radical, causing free radical damage, or its oxidative product Peroxynitrite has caused the oxidative stress in the body, the same amount of extra Citrulline will be produced, as it is connected with NO production.
By comparing Citrulline levels to other markers of Oxidative Stress and Heavy Metal levels, it is possible in broad terms to identify what the root cause of the oxidative stress in the body.
Citrulline is one of the Intermediary Metabolites and Diagnostic Markers measured in Genova Diagnostics' Amino Acid Analysis urine test and the Optimal Nutrition Evaluation (ONE) urine test (which is basically the Amino Acid Analysis plus the Metabolic Analysis Profile). Other markers for Oxidative Stress in this test are discussed below. Please see the Amino Acid Tests section on the Tests page.
It is also possible to measure Citrulline levels in the blood using a serum blood test, which perhaps provides a more 'real time' picture of NO production.
Peroxynitrite is able to act as a nitrating agent directly or indirectly through NItrogen Dioxide (NO2 and CO3- being the products of the reaction of ONOO- with CO2). When either NO2 or ONOO- nitrate the amino acid Tyrosine, the resulting product is Nitrotyrosine.
Nitrotyrosine is found in a number of pathological conditions and is considered a marker for NO-dependent oxidative stress. In Keratoconus, a degenerative eye disorder, it is found in the cornea. Nitrotyrosine is believed to partipate in the pathogenesis of diabetes. It is likely that Nitrotyrosine levels may be elevated in CFS, ME or Fibromyalgia patients.
Oxidative Stress tests
A number of tests are available to determine the presence of oxidative stress in the body, including:
8-OHdG and 8-OHG test for DNA and RNA damage by free radicals or ROS.
F-2 Alpha Isoprostane test - marker for oxidative damage of lipid component of cell membranes
Urinary Pterins Test - Neopterin, Biopterin and BH4
Mitochondrial membranes - Translocator Protein Studies
Metabolic Analysis Profile (see NOS byproduct tests above) - e.g. elevated Citric Acid and Cis-Aconitic Acid levels may indicate Iron-Sulphur protein damage by ONOO-.
These are discussed on the Oxidative Stress page and also on the Tests page. These do not deal specifically with ONOO- related oxidative stress, as free radicals can derive from the presence of heavy metal toxicity, but it is likely in many cases to be a combination of elevated ONOO- levels, decreased protective Glutathione levels and elevated heavy metal levels.
Inflammatory Stress tests
Probably the most important measure of cellular inflammation is the Urinary Pterins test (a.k.a. Urinary Neopterin Profile), that measures the levels of Neopterin, Tetrahydrobiopterin (BH4) and Biopterin, the markers of neuroinflammation and cellulalr immune system response and inflammation. This is described on the Tests page.