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.

Nitric Oxide and Peroxynitrite in Health and Disease

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 (300–302, 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 (92–94, 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.

Tetrahydrofolate and 5-methyltetrahydrofolate are folates with high antioxidant activity. Identification of the antioxidant pharmacophore

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 that “it 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, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis

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:-

Endothelial Dysfunction - Oxidative Stress, Inflammation and BH4

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.

The Hyperuricosuric Subtype of Autism, Uridine and Antipurinergic Therapy

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.

Xanthine oxidase inhibition reduces reactive nitrogen species production in COPD airways

“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 "

Potassium Bromide for Intractable Epilepsy and perhaps some Autism

Potassium Bromide has been on my to do list ever since I read a case study about Ida, a girl with epilepsy and non-verbal autism being treated at London’s Great Ormond Street Hospital 150 years ago.  Of course, the doctor not did not use the term autism, but it was obviously present.  

What I took away was not the resolution of her seizures but her behavioral change and most importantly the initiation of age-appropriate play.

Later I read a Brazilian paper called “Combined effect of bumetanide, bromide, and GABAergic agonists: An alternative treatment for intractable seizures”.

My first toe in the water in treating my son’s autism was to use Bumetanide.  That trial was successful and ever since I have looked at ways of increasing this bumetanide effect.

Bumetanide partially blocks the flow of chloride (Cl-) into neurons and over time lowers the concentration towards where it should be, in typical mature neurons.  This allows the neurotransmitter GABA to function as it should and brings back neurons into a less excitatory state and hence gives better cognitive function.

Other ideas to further lower the level of chloride included using the AE3 ion exchanger and so I proposed the possible use of Diamox.

It might also be possible to increase the expression of KCC2, the transporter that takes chloride out of neurons; this might be achieved using intranasal insulin or indeed IGF-1.

Yet another theoretical method might be to introduce bromide and allow it to compete with chloride.  We know that Br^– ions cross cellular membranes more quickly than Cl^–. So by adding bromide we should automatically reduce chloride concentration within neurons.

Medical use of Potassium Bromide

It is surprising how medicine varies so much by country.  One example is the continued use of potassium bromide (KBr) to treat childhood epilepsy in Germany, Austria and Japan.

It is currently used to treat severe forms of generalized tonic-clonic seizures, early-childhood-related Grand-Mal-seizures, and also severe myoclonic seizures during childhood.

KBr was the world’s first epilepsy drug and its use was pioneered by Sir Charles Locock in 1857.  It is still the first-line treatment for treating epilepsy in dogs, but no longer in humans.

Due to a very long half-life, it takes a month of use to reach a stable level, so in the earlier years it is likely that un-necessarily high doses (up to 6g per day) were used.  This led to side effects.  The modern dosage is 50 to 70 mg/kg in infants and toddlers, 30 to 50 mg/kg in school children and 20 to 30 mg/kg in adults.  Tolerability of bromide treatment is much improved.

It is possible to start therapy with a loading dosage to overcome the problem of the long half life, but I expect this just increases the chance of side effects.

My thought was that at a lower concentration than prevents seizures, bromide might still be effective in some autism that responds to bumetanide.  At such a dosage the side effects that occur in German epilepsy therapy might become trivial.

The main side effects are usually drowsiness (19%) and acneiform skin eruption (13%) at the 50mg/kg dosage.  I was thinking that at a quarter of this dose you might get the good without the bad.

If you have one of the many kids with autism and intractable epilepsy then you might as well follow the standard dosage and just accept the risk of some spots.  After all, the standard anti-epileptic drugs (AEDs) all have side effects and we are not just taking about spots.

Interestingly, while KBr does not interact directly with other AEDs, it is found in Germany that previously ineffective AEDs can become effective when the person is given KBr.  There are various theories to explain this.  As a result KBr look doubly useful for intractable epilepsy.  

Dravet Syndrome

KBr seems to be particularly effective in people with SCN1A-mutations suffering from Dravet syndrome.  You may recall that Professor Catterall trialed his low dose clonazepam therapy in the mouse model of Dravet syndrome.  German and Austrian clinicians have shown that KBr is highly effective in treating seizures in the human form of Dravet, while a Japanese retrospective analysis of 99 patients which found complete prevention of status epilepticus in 41.7% of patients receiving bromide.

Mode of Action

Nobody knows exactly why KBr is effective in epilepsy, but that also applies to many other AEDs.

The Brazilian view is:-

“bromide may exert antiepileptic activity not only because of its reinforcement of the Cl^– hyperpolarizing Nernst potential, but also because of its low affinity for the NKCC enzyme in comparison with Cl^– . In summary, bromide's antiepileptic effect may be divided into three parts: (1) compensation of Cl^– accumulation by means of its hyperpolarizing effect on chloride channels; (2) antagonism of chloride flow through the channels because of its competition with chloride; (3) low affinity for the NKCC enzyme”

That paper is:-

Combined effect of bumetanide, bromide, and GABAergic agonists: An alternative treatment for intractable seizures

The German view is:-

“While the exact mode of action of bromide is still unknown, the most acceptable hypothesis besides an inhibition of carbonic anhydrase is stabilization of excitable membranes through hyperpolarization of neurons. Bromide crosses cellular membranes more quickly than chloride, enhancing

GABA-activated inhibitory postsynaptic potentials and leading to hyperpolarization. Not only GABA-activated chloride channels are more permeable to bromide, but also voltage dependent channels. Studies using combined rat hippocampus-entorhinal cortex slices showed that bromide reduced or even blocked low calcium and low magnesium induced recurrent discharges, including the low magnesium induced late recurrent discharges which do not respond to most clinically used anticonvulsants. This mechanism might explain why our patients who previously did not

improve with various other antiepileptic drugs responded to treatment with bromide.

The above is from one of many good German papers on KBr :-

Bromidein Patients with SCN1A-Mutations Manifesting as Dravet Syndrome

Intractable Epilepsy

About one-third of people with epilepsy will eventually develop intractable epilepsy. This means that standard anti-epileptic drugs (AEDs) do not work well, or at all, to control the seizures.

Intractable epilepsy can have a big effect on life. People with intractable epilepsy may have trouble at work or school. They may worry a lot about when their next seizure will come. They may also have injuries that result from their seizures.

In the case of the 30+% of people with strictly define autism (SDA) and epilepsy things can get particularly difficult and depend a great deal on where you live.

In the US some children with severe autism and recurring seizures can still be collected from home by the school bus and dropped back at the end of the day.  Not only do they have qualified nurses at school to deal with any seizures but even the bus has a nurse.

I was just reading about a teenage girl in the UK who no longer attends school at all because she may have a seizure.  The irony here is that the girl has been to the county’s top children’s hospital, Great Ormond Street.  Had she been there one hundred and fifty years ago she would have been prescribed KBr.  Had she attended a hospital in Innsbruck or Salzburg, Austria this year she would very likely also have been prescribed KBr.

The literature supporting the use of KBr is published in the English language and so there is no excuse for epilepsy experts not to be aware of it. Both the US and the UK have provisions in place where clinicians can apply to treat patients with non locally approved drugs.  So there is nothing to stop a neurologist or epileptologist in the US or UK from using KBr if he really wants to.  He just has some extra paperwork.  The simpler solution if you have intractable epilepsy might be to pay a visit to Germany, Austria or indeed Japan. Or you go see the vet.

Conclusion

This blog does not have many German/Austrian readers, in fact for a condition “invented” by Austrians (Kanner and Asperger) there is very little coming out of that part of the world nowadays.

German/Austrian parents would be the ones best placed to see the effect of KBr on intractable epilepsy and perhaps some autism.

Any readers that do try potassium bromide are very welcome to share their experiences.

Quantifying the Benefits of Stimulation over Neglect in Early Childhood

Today’s post is about the Bucharest Early Intervention Project (BEIP), which really deserves a mention somewhere in any autism blog.  It has been going for many years but they recently added some very tangible MRI data.

BEIP is a long term study lead by Charles Nelson, a professor of neuroscience and psychiatry at Harvard Medical School.  It compares the effect of neglect versus stimulation in early childhood.

You may be wondering the relevance of this to autism, in particular since Kanner’s old theory about refrigerator mothers was debunked long ago.

The study shows how physical development of the brain can be altered by the living environment of a young child.  It reinforces the fact that institutionalized of young children, with or without developmental disorders, is precisely the wrong strategy.

Bucharest Early Intervention Project (BEIP)

The Bucharest Early Intervention Project was a randomized controlled trial of foster care as an intervention for children abandoned at or around the time of birth and placed in one of six institutions for young children in Bucharest, Romania.

The BEIP began in 2000 with a comprehensive baseline assessment of 136 children and their caregiving environments. Following this assessment, half the children were randomly assigned to high-quality foster care (designed specifically for this study) and the other half to remain in institutional care. The average age at entry into foster care was 22 months (range=6-31 months). All children were seen for follow-up assessments at 30, 42 and 54 months, 8 years, and 12 years, and the development of children in foster care was compared to the development of children randomized to remain in institutional care and to a group of never institutionalized children (community controls). 

Findings through the assessment at 12 years of age suggest that early institutionalization leads to profound deficits in many domains examined to date, including cognitive (i.e., IQ) and socio-emotional behaviors (i.e., attachment), brain activity and structure, alterations in reward sensitivity and processing, and a greatly elevated incidence of psychiatric disorders and impairment. 

  

The foster care intervention was broadly effective in enhancing children’s development, and for specific domains, including brain activity (EEG), attachment, language, and cognition, there appear to be sensitive periods regulating their recovery. That is, the earlier a child was placed in foster care, the better their recovery. Although the sensitive periods for recovery vary by domain, our results suggest that placement before the age of 2 years is key.

  

There are a few areas, such as executive functioning (i.e., memory and cognitive monitoring), in which placement into foster care does not significantly impact development/performance. 

In 2015 another paper was published.

BEIP initially enrolled 136 children in research. Only 69 were involved in the MRI study, of these, 23 were drawn from the group randomly assigned to foster care, 26 from a group assigned to remain in orphanages, and 20 from the local community, as controls. Lead author Johanna Bick, a clinical psychologist at the Boston Children’s Hospital, and colleagues in the BEIP group used an MRI technique called diffusion tensor imaging to look at the microstructure of 48 white matter tracts in each child, comparing results at 2 years and 8 years of age.

The analysis found that the children who stayed in orphanages were consistently worse off—with less mature development in four key sets of white matter. The most affected tracts included nerve circuits involved in general cognitive performance, emotion, maintaining attention and executive function, and sensory processing. Another analysis suggested that the foster care group was more like the community group in brain development, but this finding appears to be less robust.

Other nonrandomized studies have reported broad cognitive deficits or reduced white matter in adults and some children who suffered neglect or maltreatment in the past. They highlighted "the same regions that we find affected by early life neglect. These results and those from BEIP converge," Bick claims.

Four estimates of white matter integrity (Fractional Anisotropy, and Mean, Radial, and Axial Diffusivity) for 48 white matter tracts throughout the brain were obtained through Diffusion Tensor Imaging.

Significant associations emerged between early life neglect and microstructural integrity of the body of the corpus callosum and tracts involved in limbic circuitry (fornix crus, cingulum), fronto-striatal circuitry (anterior and superior corona radiata, external capsule) and sensory processing (medial lemniscus, retrolenticular internal capsule). Follow up analyses revealed that early intervention promoted more normative white matter development among previously neglected children who entered foster care.

Results suggest that removal from conditions of severe early life neglect and entry into a high quality family environment can support more normative trajectories of white matter growth. Findings have implications for public health and policy efforts designed to promote normative brain development among vulnerable children.

The BEIP study started ten years after the fall of communism in Romania, when the outside world became aware of life inside their orphanages.  As childless couples from the West started to adopt Romanian orphans it became clear that there was a very high prevalence of autism and other disorders.

Romania had been a country under extreme communism with a dictator, Nicolae Ceaușescu, but his official tittle he was General Secretary of the Romanian Communist Party, from 1965 to 1989.  He had some very particular ideas.  He wanted to encourage large families so both abortion and contraception were banned.  He did not like the idea of foreign debt and in his later years decided to pay down the nation’s debt as a matter of urgency.   In 1982, to be rid of foreign debt, Ceaușescu ordered the export of much of the country’s agricultural and industrial production. What followed was extreme poverty and people did not have enough food.  Excess children were deposited at the orphanage because there was no food to feed them at home.  So Romania developed a totally oversized orphanage system, that was itself extremely poorly funded.  Children were often totally neglected, left unclothed, some chained to their beds and given no stimulation.  Older children beat younger children.  All kinds of children ended up in orphanages and most had living parents.  Even after the Revolution of December 1989 which ended with Ceaușescu  and his wife being executed by firing squad, things did not improve very much, due to the dire state of the economy.  Foreigners later started to adopt children from Romania’s orphanages.

Nelson did not go to Bucharest to study autism, he went to study the consequences of neglect and to see if those consequences could be reversed.

The studies do show how a warm stimulating environment can reverse some physical brain malformations, but most effectively intervening before the age of two.

There are clear parallels with autism where some children effectively exclude themselves and when great efforts are made to engage with them using any one of a variety of therapies from Floortime, to Son Rise to ABA, great progress can sometimes be made.

When my own son was diagnosed aged 3.5 years, the developmental pediatrician told me that there was no way to predict his outcome, because up to the age of 5 years old the brain can develop remarkably.  She sees very many such children.

In the parts of the US autism diagnoses is possible before the age of 2 years old.  It would be useful if clinicians routinely carried out MRI scans of such children and the tracked their development keeping a note of what therapy the parents implemented.  Then we might see whether there were indeed defects in the microstructural integrity of the body of the corpus callosum and tracts involved in limbic circuitry and in which children these defects reduced in later years.

Monty’s new assistant was just telling me how she went to a Floortime seminar, but there was nothing much new in it and she will instead continue with he plans to study ABA.  There are so many of these therapies, the most ridiculed one is Son Rise.  My conclusion a long time ago was that it does not matter which of these “hands on” interventions you follow, you just need to be animated, energetic and engage with the child in an intensive fashion all day and every day.

A child with severe autism is trying to do the opposite, preferring to sit in a swing or watch videos all day.  This is hardly more stimulating than the Romanian “orphan” neglected in his dormitory with twenty other boys.

Just as Nelson has shown that normal babies can be made to develop brain abnormalities by their living environment and that these abnormalities can be reversed by changing their living environment, we need to know to what extent similar brain abnormalities exist in some autism and whether they are reversible, in some cases, by intense Mary Poppins-like intervention.  I suspect this is indeed the case.  If people with autism were routinely monitored this would be easy to prove one way or the other.

If the parents of the two year old, just diagnosed with autism, were told the child was in the 20% group that has some structural brain anomalies that are known to be partially reversible by some extreme Mary Poppins-like intervention, they probably would do something about it.

In fact all kids would benefit from a Mary Poppins, but perhaps some much more so than others.

Monty had his own Poppins, his full time assistant, for several years and she achieved what had seemed impossible.  I really do not believe many parents can achieve this themselves, unaided.  All day long, providing a stimulating, educative, one on one environment is a huge task. Doing it for one hour a day is not enough.