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Showing posts with label Nitrosative Stress. Show all posts
Showing posts with label Nitrosative Stress. Show all posts

Friday 2 June 2023

Nitric Oxide in Autism - nNOS as a precise target for treatment?

Today’s subject is not new to this blog, it is Nitric Oxide (NO) and how by reducing expression of the enzyme nNOS, which produces NO in neurons, you may reduce the severity of autism symptoms.  Monty has actually been reducing nNOS for several years using Agmatine.

The research is from Israel, which is better known for autism research into cannabis.

Several posts in this blog refer to NO:

https://www.epiphanyasd.com/search/label/Nitric%20Oxide

One introduces nitrosative stress, which is also covered in my book.

Nitrosative Stress, Nitric Oxide and Peroxynitrite


Nitric oxide performs many functions within the body.

I did make the graphic below a few years ago to show what happens to Arginine in the body and the role of my supplement Agmatine.

Arginine is converted to Nitric Oxide in the body by one of 3 enzymes (iNOS, eNOS and nNOS).

eNOS (endothelial nitric oxide synthase) will help expand blood vessels, lowering blood pressure and potentially boosting exercise endurance.

nNOS (neuronal nitric oxide synthase) is involved in the development of nervous system. It functions as a neurotransmitter important in long term potentiation and hence is likely to be important in memory and learning. nNOS has many other physiological functions, including regulation of cardiac function and peristalsis and sexual arousal in males and females.

iNOS (inductible nitric oxide synthase), involved in immune response, and produces NO as an immune defence mechanism, as NO is a free radical with an unpaired electron. It is the proximate cause of septic shock and may function in autoimmune disease.

 

I have used Agmatine as a supplement in my PolyPill for many years. It reduces iNOS and nNOS while increasing eNOS.

Note that you can use polyamines to induce autophagy and this idea is now used to improve cognition in people with dementia. Wheat seedlings and wheat germ are a rich source of polyamines and can simply be added to bread to make it counter some dementia.

 


Nitrosative stress

Nitrosative stress is the lesser known twin of oxidative stress. Both are generally bad for you (unless you have cancer, because cancer cells are vulnerable to it).

Nitrosative stress and oxidative stress both feature in most autism. The more severe the autism the higher the level of nitrosative stress.  Where there is nitrosative stress, expect to also see unusual amounts of NO.

Peroxynitrite from nitrosative stress can be quenched by Leucovorin, AKA calcium folinate. This is Dr Frye’s therapy for folate deficiency, but as I have mentioned previously it also has totally unrelated potential benefits. 

Now to see what the Israelis have been up to.

 

Israeli study reveals potential method for reducing symptoms of autism

Researchers find a direct link between levels of nitric oxide in the brain and condition in mice; reducing the amounts lowers indicators and behaviors. 

Researchers from the Hebrew University of Jerusalem have published a first-of-its-kind study revealing a potential future method for reducing the symptoms of autism among those diagnosed with the common developmental disorder.

Dr Haitham Amal and his team from the School of Pharmacy in the Faculty of Medicine discovered a direct connection between levels of nitric oxide (NO) in the brain and autism, the university said in a statement.

The study, conducted on mice and published Monday in the peer-reviewed Advanced Science journal, demonstrates that autism indicators increases as NO increases in the brain, and that autism indicators and behavior decrease as the levels of NO in the brains of murine models of autism are lowered “in a proactive and controlled manner.” 

 

“Our research showed – in an extraordinary way – that inhibiting the production of NO, specifically in brain neuron cells in mouse models of autism, causes a decrease in autism-like symptoms,” he said. “By inhibiting the production of NO on laboratory animals, they became more ‘social’ and less repetitiveness was observed in their behavior. Additionally, the animals showed interest in new objects and were less anxious. Finally, the decrease in NO levels led to a significant improvement in neuronal indices.”

 

Scientists identify a new molecular mechanism for autism - Advanced Science News

 

After having tested their hypothesis in living mice, the researchers turned their focus to cell cultures. To begin with, they cultured neuronal cells from normal and mutant mouse models. Increasing and decreasing levels of nitric oxide in these cultures led to similar biochemical changes as those seen in experiments with mice.

Having investigated the impact of nitric oxide in mice, Amal’s team sought to confirm their findings in humans. First, they tested neurons that were derived from the stem cells of people with mutations in the SHANK3 gene, living with ASD. These neurons had high levels of proteins that help diagnose stress caused by nitric oxide. When researchers treated these neurons with a nitric oxide inhibitor, the levels of these proteins subsided.

Thereafter, Amal’s lab measured the levels of the same proteins in samples of blood plasma taken from children with ASD. They wanted to validate their results in this demographic. Compared with unaffected children, those with ASD had higher levels of biomarkers that indicate nitric oxide stress.

Deeper analyses revealed that the production of numerous proteins responsible for neuronal development was increased or decreased, differing from their normal levels. Further, using computational analyses, the researchers found that genes involved in several mechanisms connected to ASD development were overrepresented. These genes are key to severing connections between neurons as well as driving inflammation and oxidative stress.

“This research is a significant breakthrough in autism research with the first direct connection made between an increase in the concentration of [nitric oxide] in the brain and autistic behavior,” said Amal. “I am hopeful that with our new understanding of the [nitric oxide] mechanism, we can begin to develop therapeutic drugs for ASD and help millions of children and adults living with autism around the world.”

Amal’s team is exploring the impact of nitric oxide in many more models of autism. “The good news is that we are exploring very similar data,” added Amal.

 

 

The NO Answer for Autism Spectrum Disorder

Autism spectrum disorders (ASDs) include a wide range of neurodevelopmental disorders. Several reports showed that mutations in different high-risk ASD genes lead to ASD. However, the underlying molecular mechanisms have not been deciphered. Recently, they reported a dramatic increase in nitric oxide (NO) levels in ASD mouse models. Here, they conducted a multidisciplinary study to investigate the role of NO in ASD. High levels of nitrosative stress biomarkers are found in both the Shank3 and Cntnap2 ASD mouse models. Pharmacological intervention with a neuronal NO synthase (nNOS) inhibitor in both models led to a reversal of the molecular, synaptic, and behavioral ASD-associated phenotypes. Importantly, treating iPSC-derived cortical neurons from patients with SHANK3 mutation with the nNOS inhibitor showed similar therapeutic effects. Clinically, they found a significant increase in nitrosative stress biomarkers in the plasma of low-functioning ASD patients. Bioinformatics of the SNO-proteome revealed that the complement system is enriched in ASD. This novel work reveals, for the first time, that NO plays a significant role in ASD. Their important findings will open novel directions to examine NO in diverse mutations on the spectrum as well as in other neurodevelopmental disorders. Finally, it suggests a novel strategy for effectively treating ASD.

 


 

NO Donor Administration Induced ASD-Like Behavior in WT Mice and Enhanced the ASD Phenotype in Mutant Mice 

NO Inhibition Reversed Synaptophysin Expression and Reduced Nitrosative Stress in Primary Cortical Neurons Derived from the Mutant Mouse Model 

nNOS Inhibition Restores the Expression of Key Synaptic Proteins Using iPSC-Derived Cortical Neurons from Patients with SHANK3 Mutations

Elevation of Nitrosative Stress Biomarker and Reprogramming of the SNO-Proteome in the Blood Samples of ASD Children

 

Our study is designed to examine the effect of high levels of NO on the development of ASD. This work shows that NO plays a key role in ASD. Importantly, this was confirmed in cellular, animal models, human iPSC-derived cortical neurons, as well as in clinical samples. Since the molecular mechanisms underlying ASD pathogenesis remain largely unknown, we provided a new mechanism that shows that NO plays a key role in ASD pathology at the molecular, cellular, and behavioral levels. An increase of Ca2+ influx in ASD pathology, including in human and mouse models of Shank3 and Cntnap2(-/-), has already been reported. Ca2+ activates nNOS, which then leads to massive production of NOAberrant NO production induces oxidative and nitrosative stress, leading to increased 3-Ntyr production and aberrant protein SNO. Our data showed an increase in NO metabolites and 3-Ntyr production in both mouse models of ASD (Shank3Δ4-22, Cntnap2(-/-)). Increased 3-Ntyr was found in iPSC-derived cortical neurons from patients with SHANK3 mutations, SHANK3 knocked down in SHSY5Y cells, and in human ASD plasma samples. The elevated levels of 3-Ntyr in our study are consistent with previous postmortem examinations of ASD patients showing the accumulation of this molecule in the brain. 

Collectively, our results show for the first time that NO plays a key role in ASD development. We found that NO affects synaptogenesis as well as the glutamatergic and GABAergic systems in the cortex and the striatum, which converge into ASD-like behavioral deficits. This work suggests that NO is an important pathological factor in ASD. Examining NO in diverse mutations on the spectrum as well as other neurodevelopmental disorders and psychiatric diseases will open novel future research directions. Finally, this is a novel experimental study that establishes a direct link between NO and ASD, leading to the discovery of novel NO-related drug targets for the disorder and suggesting nNOS as a precise target for treatment.

 

The trigger for the excess NO production is put down to the increase of Ca2+ influx, which really is at the core of autism.  This was explained in the post about IP3R long ago. 

Is dysregulated IP3R calcium signaling a nexus where genes altered in ASD converge to exert their deleterious effect?

 

The simple answer appears to be YES.

 and in later posts:

https://www.epiphanyasd.com/search/label/IP3R

  

Conclusion

For autism a little less nNOS, please.

The researchers used the selective neuronal nitric oxide synthase inhibitor 7-nitroindazole.

Nitroindazole acts as a selective inhibitor for neuronal nitric oxide synthase, an enzyme in neuronal tissue, that converts arginine to citrulline and nitric oxide (NO).

7-Nitroindazole is under investigation as a possible protective agent against nerve damage caused by excitotoxicity or neurodegenerative diseases. It may act by reducing oxidative stress or by decreasing the amount of peroxynitrite formed in these tissues. These effects are related to the inhibition of type 1 nitric oxide synthase. However, anti-convulsive effect is derived from some other mechanisms. 

For older folks with higher blood pressure, a little more eNOS please; indeed, the explosive nitroglycerin is also used as a life-saving drug that induces eNOS production in someone about have a heart attack. The resulting NO widens blood vessels and so increases blood flow.


Methylene blue was mentioned in a recent comment in regard to nitric oxide (NO)

Methylene blue (MB) inhibits endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), guanylate cyclase, and cytokines such as tumor necrosis factor-α (TNF-α). MB restores vascular tone due to the selective blockade of both guanylate and iNOS.

MB should increase blood pressure.

Some people with autism respond well to MB. This likely is unrelated to its effect on NO and might well be due to its numerous anti-inflammatory effects (inhibiting NLRP3 inflammasome etc).










 

Thursday 8 December 2016

Nitrosative Stress, Nitric Oxide and Peroxynitrite










In this example of Brain Injury, developing oligodendrocytes are injured and killed by substances released from activated microglia, including nitric oxide and superoxide, which form peroxynitrite. Peroxynitrite has been found to kill these cells through the activation of the 12-lipoxygenase pathway for metabolizing arachidonic acid. Mitochondria may be involved in this pathway as a source of reactive oxygen species.


Much has been written in this blog about oxidative stress, which has now been extremely well researched in autism and more generally. Let’s recap oxidative stress.

The most knowledgeable researcher in this area is Abha Chauhan.  Based on her research and that of others we now know a great deal.  Recall that the body’s key antioxidant is called glutathione (GSH) and when it neutralizes a free radical GSH is converted to its oxidized form, glutathione disulfide (GSSG).  A good measure of oxidative stress is the ratio of  GSH/GSSG.


·        Autism is associated with deficits in glutathione antioxidant defence in selective regions of the brain.

·        In the cerebellum and temporal cortex from subjects with autism, GSH levels are significantly decreased by 34.2 and 44.6 %, with a concomitant increase in the levels of GSSG

·        There is also a significant decrease in the levels of total GSH (tGSH) by 32.9 % in the cerebellum, and by 43.1 % in the temporal cortex of subjects with autism.

·        In contrast, there was no significant change in GSH, GSSG and tGSH levels in the frontal, parietal and occipital cortices in autism

·        The redox ratio of GSH to GSSG was also significantly decreased by 52.8 % in the cerebellum and by 60.8 % in the temporal cortex of subjects with autism, suggesting glutathione redox imbalance in the brain of individuals with autism.

·        Disturbances in brain glutathione homeostasis may contribute to oxidative stress, immune dysfunction and apoptosis, particularly in the cerebellum and temporal lobe, and may lead to neurodevelopmental abnormalities in autism.


·        The activity of glutathione cysteine ligase (GCL), an enzyme for glutathione synthesis is impaired in autism.

·        The protein expression of its modulatory subunit GCLM was decreased in autism.

·        The activities of glutathione peroxidase (GPx) and glutathione S-transferase were decreased in autism.



For those interested, GPx is a family of enzymes that catalyze the reaction that converts GSH to GCCG.  So in order to soak up those free radicals you need both GSH and GPx.

Glutathione cysteine ligase (GCL) is a key enzyme needed to make the antioxidant GSH.  Dysregulation of GCL enzymatic function and activity is known to be involved in many human diseases, such as diabetes, Parkinson's disease, Alzheimer’s disease, COPD, HIV/AIDS, cancer and autism.  Without sufficient GCL your body cannot make enough glutathione (GSH).


I did have some conversation with Abha Chauhan a few years ago when I found that NAC (N-acetyl cysteine), a known precursor to GSH, really does have a positive behavioral impact in autism.  She is clearly very nice, but not the type to make the leap to translating her science into therapy.

As I have shown there are many other treatable aspects of oxidative stress.

The chart below is my annotated version of the original by Professor Helmut Sies, the German “Redox Pioneer”.  He has published 500 scientific papers.




Nitrosative Stress


Finally to nitrogen.

Nitrogen is the most common pure element in the earth, making up 78.1% of the entire volume of the atmosphere.  Although nitrogen is non-toxic, when released into an enclosed space it can displace oxygen, and therefore presents an asphyxiation hazard. 

Nitrogen is an anesthetic agent. Nitrous oxide (N2O) is commonly known as laughing gas.  It is used in medicine for its unaesthetic and analgesic effects

It is also used as an oxidizer in rocket propellants, and in motor racing to increase the power output of engines, like Mad Max.

In humans we are dealing with Nitric Oxide (NO) and when things go wrong with peroxynitrite and then other Reactive nitrogen species (RNS).  In simple terms Reactive nitrogen species (RNS), like Reactive oxygen species (ROS) are bad news.

Nitric Oxide (NO) itself does lots of good things in your body.  Too much may not be good, but a little more can actually do you good.  NO is a potent vasodilator.

For over 130 years, nitroglycerin has been used to treat heart conditions, such as angina and chronic heart failure.  Nitroglycerin produces nitric oxide (NO). In hospital most patients will receive nitroglycerin during and after a heart attack, people at risk of a heart attack often carry nitroglycerin with them.

If you want to lower your blood pressure or an athlete wanting to legally improve exercise endurance you can increase Nitric Oxide (NO) via diet.  One easy way is to drink beetroot juice, as is common in endurance cycling.  In people with peroxynitrite-derived radicals this may be unwise, because they may have too much NO.



Peroxynitrite

The starting point for the production of those unhelpful Reactive Nitrogen Species (RNS) is this chemical reaction



NO (nitric oxide) + O2· (superoxide) → ONOO (peroxynitrite)



NO production is affected by the enzyme nitric oxide synthase 2 (NOS2).

Superoxide production is catalyzed by NADPH oxidase.

Superoxide also produces Reactive Oxygen Species (ROS).

NADPH oxidase is implicated in many diseases including schizophrenia and autism.

NADPH oxidase 4 (Nox4) activity decreases mitochondrial function (chain complex I).

Activated microglia (as found in autism) produce both nitric oxide and superoxide and are therefore a source of peroxynitrite.




This has started to get rather complicated. So those interested in NADPH should refer to the literature.


Peroxynitrite can directly react with various biological targets and components of the cell including lipids, thiols, amino acid residues, DNA bases, and low-molecular weight antioxidants.


Additionally peroxynitrite can react with other molecules to form additional types of RNS including nitrogen dioxide (·NO2) and dinitrogen trioxide (N2O3) as well as other types of chemically reactive free radicals.



Nitric Oxide and Peroxynitrite in Health and Disease

I have referred on this blog to Abha Chauhan’s mammoth book on oxidative stress in autism on several occasions.  A work of similar quality but this time on Nitric Oxide and Peroxynitrite, is the paper below, by Hungarian Pal Pacher, who works at the US National Institute of Health’s Section on Oxidative Stress Tissue Injury.  He looks like a citation generating machine.

You could spend a long time reading this paper, but in summary peroxynitrite and its derived products have a negative effect on a very wide range of conditions including all the common neurological conditions, inflammatory diseases and again diabetes.  The answer would be peroxynitrite scavengers.



The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.


Some excerpts:-


·        The different events set in motion by the initial generation of peroxynitrite indicate that potent peroxynitrite decomposition catalysts and PARP inhibitors might represent useful therapeutic agents for debilitating chronic inflammatory diseases

·        In summary, available evidence indicates that NO plays dichotomous roles (promotion vs. suppression) in tumor initiation and progression. The activation of angiogenesis and the induction of DNA mutations represent key aspects of the procarcinogenic effects of NO. Peroxynitrite is emerging as a major NO-derived species responsible for DNA damage, mainly through guanine modifications and the inhibition of DNA repair enzymes. In chronic inflammatory states, the identification of 8-nitroguanine in tissues indicates that nitrative DNA damage consecutive to overproduction of NO and peroxynitrite may represent an essential link between inflammation and carcinogenesis.

·        In summary, the different studies listed above indicate that small amounts of NO produced by eNOS in the vasculature during the early phase of brain ischemia are essential to limit the extent of cerebral damage, whereas higher concentrations of NO, generated initially by nNOS and later by iNOS, exert essentially neurotoxic effects in the ischemic brain. Evidence that such toxicity depends, in large part, on the rapid reaction of NO with locally produced superoxide to generate peroxynitrite will be now exposed
  

·        NO is produced by all brain cells including neurons, endothelial cells, and glial cells (astrocytes, oligodendrocytes, and microglia) by Ca2+/calmodulin-dependent NOS isoforms. Physiologically NOS in neurons (nNOS, type I NOS) and endothelial cells (eNOS, type III NOS) produce nanomolar amounts of NO for short periods in response to transient increases in intracellular Ca2+, which is essential for the control of cerebral blood flow and neurotransmission and is involved in synaptic plasticity, modulation of neuroendocrine functions, memory formation, and behavioral activity (491, 890, 1229). The brain produces more NO for signal transduction than the rest of the body combined, and its synthesis is induced by excitatory stimuli. Consequently, NO plays an important role in amplifying toxicity in the CNS. Indeed, under various pathological conditions associated with inflammation (e.g., neurodegenerative disorders and cerebral ischemia), large amounts of NO are produced in the brain as a result of the induced expression of iNOS (type II NOS) in glial cells, phagocytes, and vascular cells, which can exert various deleterious roles (39, 491, 890). Thus NO may be a double-edged sword, exerting protective effects by modulating numerous physiological processes and complex immunological functions in the CNS on one hand and on the other hand mediating tissue damage (446, 491, 890). The detailed discussion of the role of NO in the pathophysiology of various neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), just mentioning a few, is the subject of numerous excellent recent overviews (77, 145, 194, 219, 491, 890, 1003, 1205, 1433) and beyond the scope of this paper. Here we cover only the role of peroxynitrite and protein nitration, which are likely responsible for most deleterious effects of NO in neurodegenerative disorders.


·        Peroxynitrite formation has been implicated in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, MS, ALS, and traumatic brain injury (reviewed in Refs. 194, 608, 1119, 1284). Nitrotyrosine immunoreactivity has been found in early stages of all of these diseases in human autopsy samples as well as in experimental animal models. Increased nitrite, nitrate, and free nitrotyrosine has been found to be present in the cerebral spinal fluid (CSF) and proposed to be useful marker of neurodegeneration (168; reviewed in Refs. 608, 1119, 1284). Once formed in the diseased brain, peroxynitrite may exert its toxic effects through multiple mechanisms, including lipid peroxidation, mitochondrial damage, protein nitration and oxidation, depletion of antioxidant reserves (especially glutathione), activation or inhibition of various signaling pathways, and DNA damage followed by the activation of the nuclear enzyme PARP (608, 1119, 1284).


·        Uric acid has proven to be a useful inhibitor of tyrosine nitration (although it is not a direct peroxynitrite scavenger) (1271) and has been shown to protect the blood-brain barrier and largely prevent the entry of inflammatory cells into the CNS (566, 567). Additionaly, it also prevented CNS injury after inflammatory cells have already migrated into the CNS (1141). Urate has also proven beneficial in reducing the morbidity associated with viral infections (710, 1141). Interestingly, in humans there is an inverse correlation between affliction with gout and MS (710, 1195). Numerous studies have reported lower levels of uric acid in MS patients favoring the view that reduced uric acid in MS is secondary to its “peroxynitrite scavenging” activity during inflammatory disease, rather than a primary deficiency (reviewed in Ref. 694). These studies have also suggested that serum uric acid levels could be used as biomarkers for monitoring disease activity in MS

  

·        Recent evidence suggests that mitochondrial complex I inhibition may be the central cause of sporadic PD and that derangements in complex I lead to α-synuclein aggregation, which contributes to the demise of dopamine neurons (293). Accumulation and aggregation of α-synuclein may further facilitate the death of dopamine neurons through impairments in protein handling and detoxification (293). As already mentioned above, both mitochondrial complex I and synuclein can be targets for peroxynitrite-induced protein nitration


·        The significance of this intricate interplay may have important ramifications not only for ALS but also for PD and AD (6, 58, 1102). Reactive astrocytes are common hallmark of neurodegeneration, and these results suggest that peroxynitrite may play an important role in promoting this phenotype as well as causing the degeneration of neurons. In ALS, the transformation of astrocytes into a reactive phenotype may explain why ALS is progressive, causing the relentless death of neighboring motor neurons. Interfering in such a cascade to stop the progressive death of motor neurons would not necessarily cure ALS but may keep it from being a death sentence.


·        There is accumulating evidence suggesting that increased oxidative stress and excessive production of NO might contribute to the development of HD by damaging neighboring neurons (reviewed in Refs. 63, 163). Accordingly, increased iNOS expression was observed in neuronal, glial, and vascular cells from brains of HD patients and mouse models of disease (206, 491). Similarly, numerous studies have demonstrated increased 3-NT formation in brain tissues (neurons, glia, and/or vasculature) of mice transgenic for the HD mutation, rats injected into the striatum with quinolinic acid (rat model of HD), and HD patients (300302, 427, 1022, 1023, 1096, 1117). Importantly, both NOS inhibitors and peroxynitrite scavengers decreased neuronal damage, improved disease progression, and also decreased brain 3-NT content in experimental models (301, 1022, 1117). These results suggest that peroxynitrite might be an important mediator of oxidative damage associated with HD.


·        The pathogenetic role of peroxynitrite in TBI is supported by evidence demonstrating increased brain 3-NT levels following TBI in experimental mouse and rat models (9294, 423, 507, 508, 898, 1171, 1360), and by the beneficial effects of NOS inhibitor and peroxynitrite scavengers in reducing neuronal injury and improving neurological recovery following injury (423, 508, 898).Collectively, multiple lines of evidence discussed above provide strong support for the important role of peroxynitrite formation and/or protein nitration in neurodegenerative disorders and suggest that the neutralization of this reactive species may offer significant therapeutic benefits in patients suffering from these devastating diseases.


·        Collectively, the evidence reviewed above support the view that peroxyntrite-induced damage plays an important role in numerous interconnected aspects of the pathogenesis of diabetes and diabetic complications. Neutralization of RNS or inhibition of downstream effector pathways including PARP activation may represent a promising strategy for the prevention or reversal of diabetic complications.

·        In conclusion, multiple lines of evidence discussed above and listed in Table 4 suggest that peroxynitrite plays an important role in various forms of cardiovascular dysfunction and injury; pharmacological neutralization of this reactive oxidant or targeting the downstream effector pathways may represent a promising strategy to treat various cardiovascular disorders.


·        In summary, circulatory shock is a leading cause of death in intensive care units. Considerable improvement in our understanding of the molecular and cellular mechanisms of shock over the past 20 years makes it now a reasonable expectation that novel, efficient mechanism-based therapies will emerge in the near future. Considerable evidence now exists that overproduction of NO and superoxide, triggering the generation of large amounts of peroxynitrite, is a central aspect of shock pathophysiology. In addition to direct cytotoxic effects such as the peroxidation of lipids, proteins, and DNA, peroxynitrite also occupies a critical position in a positive feedback loop of inflammatory injury, by (directly or indirectly, via PARP activation) activating proinflammatory signaling and by triggering the recruitment of phagocytes within injured tissues, leading to further NO, superoxide, and peroxynitrite production, which will progressively amplify the initial inflammatory reactions (see sect. VID, Fig. 14). These various observations support the view that future strategies reducing peroxynitrite or its precursors might have a considerable therapeutic impact in clinical circulatory shock.


Peroxynitrite Scavengers


We have already covered two substances in this blog that are potential Peroxynitrite Scavengers:-


Calcium Folinate

This is Roger’s magic pill to treat his Cerebral Folate Deficiency, but it may have application far beyond this, likely rare, condition, for those that tolerate it.

Tetrahydrofolic acid, or tetrahydrofolate, is a folic acid derivative. It has the potential to quench those peroxynitrite-derived radicals.




The presumed protective effect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating effect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating effect is probably important for the antioxidant activity of THF and 5-MTHF.


Uric Acid

Uric acid has proven to be a useful inhibitor of tyrosine nitration.  It was thought to be a scavenger of peroxynitrite, but our clever Pal from Hungary tells thatit is not a direct peroxynitrite scavenger ….Numerous studies have reported lower levels of uric acid in MS patients favoring the view that reduced uric acid in MS is secondary to its “peroxynitrite scavenging” activity during inflammatory disease, rather than a primary deficiency”.

An old paper:-



Uric acid, the naturally occurring product of purine metabolism, is a strong peroxynitrite scavenger, as demonstrated by the capacity to bind peroxynitrite but not nitric oxide (NO) produced by lipopolysaccharide-stimulated cells of a mouse monocyte line. In this study, we used uric acid to treat experimental allergic encephalomyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations. Uric acid administration was found to have strong therapeutic effects in a dose-dependent fashion. A regimen of four daily doses of 500 mg/kg uric acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared. The requirement for multiple doses is likely to be caused by the rapid clearance of uric acid in mice which, unlike humans, metabolize uric acid a step further to allantoin. Uric acid treatment also was found to diminish clinical signs of a disease resembling EAE in interferon-γ receptor knockout mice. A possible association between multiple sclerosis (MS), the disease on which EAE is modeled, and uric acid is supported by the finding that patients with MS have significantly lower levels of serum uric acid than controls. In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and gout (hyperuricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that hyperuricemia may protect against MS.



Here we have a paper with the link to Tetrahydrobiopterin (BH4,), also known as sapropterin, covered in an old post:-




Interactions of peroxynitrite with uric acid in the presence of ascorbate and thiols: Implications for uncoupling endothelial nitric oxide synthase

It has been suggested that uric acid acts as a peroxynitrite scavenger although it may also stimulate lipid peroxidation. To gain insight into how uric acid may act as an antioxidant, we used electron spin resonance to study the reaction of uric acid and plasma antioxidants with ONOO-. Peroxynitrite reacted with typical plasma concentrations of urate 16-fold faster than with ascorbate and 3-fold faster than cysteine. Xanthine but not other purine-analogs also reacted with peroxynitrite. The reaction between ONOO- and urate produced a carbon-centered free radical, which was inhibited by either ascorbate or cysteine. Moreover, scavenging of ONOO- by urate was significantly increased in the presence of ascorbate and cysteine. An important effect of ONOO- is oxidation of tetrahydrobiopterin, leading to uncoupling of nitric oxide synthase. The protection of eNOS function by urate, ascorbate and thiols in ONOO(-)-treated bovine aortic endothelial cells (BAECs) was, therefore, investigated by measuring superoxide and NO using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) and the NO-spin trap Fe[DETC]2. Peroxynitrite increased superoxide and decreased NO production by eNOS indicating eNOS uncoupling. Urate partially prevented this effect of ONOO- while treatment of BAECs with the combination of either urate with ascorbate or urate with cysteine completely prevented eNOS uncoupling caused by ONOO-. We conclude that the reducing and acidic properties of urate are important in effective scavenging of peroxynitrite and that cysteine and ascorbate markedly augment urate's antioxidant effect by reducing urate-derived radicals.


Xanthine oxidase (XO, sometimes 'XAO') is a form of xanthine oxidoreductase, a type of enzyme that generates reactive oxygen species.[2] These enzymes catalyze the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid. These enzymes play an important role in the catabolism of purines in some species, including humans.





Because xanthine oxidase is a metabolic pathway for uric acid formation, the xanthine oxidase inhibitor allopurinol is used in the treatment of gout.


Inhibition of xanthine oxidase has been proposed as a mechanism for improving cardiovascular health.  A study found that patients with chronic obstructive pulmonary disease (COPD) had a decrease in oxidative stress, including glutathione oxidation and lipid peroxidation, when xanthine oxidase was inhibited using allopurinol.


Reactive nitrogen species, such as peroxynitrite that xanthine oxidase can form, have been found to react with DNA, proteins, and cells, causing cellular damage or even toxicity. Reactive nitrogen signaling, coupled with reactive oxygen species, have been found to be a central part of myocardial and vascular function, explaining why xanthine oxidase is being researched for links to cardiovascular health.


We also should recall that abnormalities are common in autism.





So perhaps allopurinol for those with too much uric acid?  Perhaps this is a good marker for peroxynitrites ?





Conclusion

As is often the case there some contradiction in the science.  Is NO good for you or not?  Are both high and low uric acid actually indicating the same biological problem.

It looks like the research into very expensive BH4 therapy might be better directed into peroxynitrite scavengers.

I think we have found the reason why so many people with autism respond to Leucovorin (calcium folinate) and, unlike in our friend Roger, it may not be because of cerebral folate deficiency.

It looks like many other chronic conditions from diabetes to COPD to schizophrenia might also benefit from  calcium folinate.

Before I forget, in the Helmut Sies oxidative stress graphic I did highlight selenium.  The GPx enzymes contain selenium and if there is selenium deficiency the body's key antioxidant mechanism will be compromised. According to Abha Chauhan's book,  "Likewise, levels of exogenous antioxidants were also found to be reduced in autism, including vitamin C, vitamin E, and vitamin A in plasma, and zinc and selenium in erythrocytes (James et al., 2004)".  This might suggest adding a little extra selenium.

I think Allopurinol is worth a look for some autism.  Allopurinol does indeed reduce reactive nitrogen species in COPD (severe asthma), as suggested above.



“These results suggest that oral administration of the xanthine oxidase inhibitor allopurinol reduces airway reactive nitrogen species production in chronic obstructive pulmonary disease subjects. This intervention may be useful in the future management of chronic "