Nitric Oxide (NO), Arginase and Endothelial Dysfunction in Autism

Endothelial dysfunction is not something people associate with autism. It is something I have covered previously in this blog and if you search on Google my post is about all you will find.

Endothelial dysfunction is acknowledged to be very important in diabetes, which is characterized by ROS (reactive oxygen species), reduced NO (nitric oxide) , reduced eNOS (endothelial nitric oxide synthase) and too much Arginase. There is also Peroxynitrite (ONOO⁻), an ion we have encountered before.

In autism we do already know from the research that VEGF (Vascular endothelial growth factor) is disturbed and there will be a post on that.

So when you put it all this together, it is odd that nobody has researched endothelial dysfunction and autism.  When I find something like this, my fallback is always schizophrenia. What about Endothelial Dysfunction and Schizophrenia? Sure enough, there is plenty of research on the subject, like this paper.

Microvascular dysfunction in schizophrenia: a case–control study

We tested the hypothesis that subjects with schizophrenia have impaired endothelial function.

Our findings suggest that a diagnosis of schizophrenia is associated with impaired microvascular function as indicated by lower values of VTI, irrespective of many other clinical characteristics. It might be an early indicator of cardiovascular risk in schizophrenia, and might help to identify high-risk individuals.

Endothelial Dysfunction does ultimately cause all kinds of problems in later life.  What is relevant to this blog is the potential neurological benefit of improving endothelial function in younger people, if any.

We need to recall that historically there have been very few older people with more severe autism; they did not live to the age when typical problems caused by endothelial dysfunction become apparent. 

Overlapping causes of Endothelial Dysfunction

The interesting question is just how many of the possible causes of endothelial dysfunction occur in autism. 

So far the following factors seem to apply to autism:-

·        Oxidative stress (ROS)

·        Reduced eNOS and nitic oxide (NO)    

·         VEGF (Vascular endothelial growth factor) is disturbed

·         Even estrogen deficiency can play a role and this is reduced in autism

People with autism who use calcium folinate (Leucoverin) are already quenching  Peroxynitrite (ONOO⁻) another factor in Endothelial Dysfunction.                                                               

Is Arginine/Arginase disturbed in Autism?

One well known anomaly in diabetes is a high level of an enzyme called Arginase, resulting in reduced production of nitric oxide (NO) in endothelial cells.

Here again we have to revert to looking at schizophrenia, as the closest thing to autism. Here there are no surprises. 

Altered brain arginine metabolism in schizophrenia.

Previous research implicates altered metabolism of l-arginine, a versatile amino acid with a number of bioactive metabolites, in the pathogenesis of schizophrenia. The present study, for we believe the first time, systematically compared the metabolic profile of l-arginine in the frontal cortex (Brodmann's area 8) obtained post-mortem from schizophrenic individuals and age- and gender-matched non-psychiatric controls (n=20 per group). The enzyme assays revealed no change in total nitric oxide synthase (NOS) activity, but significantly increased arginase activity in the schizophrenia group. Western blot showed reduced endothelial NOS protein expression and increased arginase II protein level in the disease group. High-performance liquid chromatography and liquid chromatography/mass spectrometric assays confirmed significantly reduced levels of γ-aminobutyric acid (GABA), but increased agmatine concentration and glutamate/GABA ratio in the schizophrenia cases. Regression analysis indicated positive correlations between arginase activity and the age of disease onset and between l-ornithine level and the duration of illness. Moreover, cluster analyses revealed that l-arginine and its main metabolites l-citrulline, l-ornithine and agmatine formed distinct groups, which were altered in the schizophrenia group. The present study provides further evidence of altered brain arginine metabolism in schizophrenia, which enhances our understanding of the pathogenesis of schizophrenia and may lead to the future development of novel preventions and/or therapeutics for the disease

Arginine metabolic pathways. l-arginine can be metabolized by NOS, arginase and ADC to form a number of bioactive molecules (see the Introduction for detailed description). We found increased levels of arginase activity, arginase II protein expression and agmatine tissue concentration (indicated by the red letters and arrows), and reduced eNOS protein expression and GABA level (indicated by the green letters and arrows) in the schizophrenia cases. ADC, arginine decarboxylase; BA8, Brodmann's area 8; eNOS, endothelial NOS; GABA, γ-aminobutyric acid; iNOS, inducible NOS; NO, nitric oxide; NOS, nitric oxide synthase; nNOS, neuronal NOS.  

It is of interest to note that the plasma agmatine level was increased over threefold in schizophrenic patients relative to healthy controls

The present study, interestingly, found an over 50% increase in arginase activity in BA8 accompanied by a significant upregulation of arginase II in the schizophrenia group. It is currently unclear how arginase changes in blood correlate with those in the brain tissue

Polyamines and agmatine have also been implicated in psychiatric disorders

L-Norvaline for Aspie1983?

One reader has raised issue of whether L-Norvaline would be a good idea.

L-Norvaline is an arginase inhibitor, used by body builders to increase nitric oxide.

L-arginine is used as a substrate by both nitric oxide synthase (NOS) and arginase to produce nitric oxide (NO) and urea, respectively.

If you inhibit arginase you shift the L-arginine over towards nitric oxide production.

People with diabetes and, as we saw above schizophrenia, have elevated levels of arginase. This will cause them to have a reduced level of nitric oxide. Reduced nitric oxide will contribute to Endothelial Dysfunction.

So it looks like L-Norvaline might well be beneficial in diabetes and schizophrenia.

L-Norvaline  might slow the conversion of ammonia to urea, if arginase was low to start with.  If arginase was elevated to start with you might expect no impact on the conversion of ammonia to urea.

Agmatine, Polyamines & L-citrulline 

Agmatine may already be elevated in schizophrenia, but it looks like a little extra can be beneficial in autism.

Polyamines can also be good for you if they increase autophagy. Which specific polyamine you want is an open question.

In the schizophrenia study L-citrulline is reduced. This makes sense because L-arginine has been shifted over towards  urea by elevated arginase. L-citrulline is a byproduct when nitric oxide (NO) is produced.

Perhaps unexpectedly, l-Citrulline is also a potent endogenous precursor of l-arginine. In a recent clinical study, l-citrulline supplementation dose-dependently increases plasma l-arginine levels in healthy human volunteers more effectively than equivalent doses of l-arginine itself.

Aspie1983 might not need to supplement L-citrulline, if he used L-Norvaline .

Altered brain arginine metabolism in autism?

I suspect Aspie1983 is not the only one with an altered brain arginine metabolism.

There appear to be many therapeutic options and they are all body building supplements because they will all increase nitric oxide (NO).

They will all improve Endothelial Dysfunction, which was the original subject of this post.

Conclusion

It certainly seems like Endothelial Dysfunction is present in some autism and that numerous established therapies should help.

We are already targeting oxidative stress with antioxidants and some people use calcium folinate that will target nitrosative stress.

The therapies that increase NO and/or eNOS include:-

·        Agmatine

·        L-arginine

·        L-citrulline

·        L- norvaline

·        Cocoa flavanols

·        Beetroot juice

·        L-taurine does increase eNOS and NO, but it is not clear how

There are products sold to body builders that include several of these and some clever additional ones.

Like this one, 12 grams made up of:-

1.   L-Citrulline

2.   L-Taurine

3.   Agmatine Sulfate 

4.   Glycerol Monostearate

5.   Dan-Shen, a Chinese cardio-protective herb that increases NO and also behaves like low dose aspirin

6.   Beetroot Powder

7.   L-Norvaline

8.   Hesperidin, a citrus flavonoid that increases NO

9.   Black pepper extract; piperine is known to affect NO release

Dan-Shen :- there are numerous clinical trials on Dan-Shen and its active ingredient. It has even been suggested to treat PANS/PANDAS.

These clinical trials include treating altitude sickness.

Hesperiden is found in oranges and indeed peppermint, but in oranges it is most abundant in the white inner part of the peel. Orange peel is a home remedy to lower cholesterol. Research shows that Hesperiden (and naringin) is a potent cholesterol lowering substance.

You would think that you can have too much of a good thing, that is too much endothelial nitric oxide; ask a body builder.

There is more to this subject, beyond the body builder’s science; the related areas to look at are angiogenesis and lymphangiogenesis. These are very much influenced by VEGF (Vascular Endothelial Growth Factor). In the next post we will see that there is evidence suggesting blood vessel growth can be unchecked in some autism resulting in unstable blood flow, not simply reduced flow.

So while the view from today’s post is that in autism there may be restricted blood flow, rather like in vascular dementia, the real situation is likely more complex.

We also have the issue of how the lymphatic system, that collects waste materials from the body (including the brain), may also be affected. With blood vessels there may be “too much growth” but in the case of lymphatic system there may be too little. This is all governed by VEGF.

We have already seen that autophagy and mitophagy are reduced in some autism and are a defining feature of Huntingdon’s Disease. Accumulation of waste products in the brain has consequences. Improved autophagy, possible via the same polyamines referred to in the earlier graphic, and improved lymphangiogenesis could be therapeutic. It appears that the brain flushes out waste products to the lymphatic system while you sleep; Alzheimer's is most prevalent in people who sleep very little.

Hypoperfusion in Autism Revisited

One old post from this blog has been going viral recently (3,000 views in one day, via Facebook) and it is quite relevant to a debate that has been going on in the comments about the potential merits and mechanisms of Hyperbaric Oxygen Therapy (HBOT). Two commenters are big fans of HBOT.

Hypoperfusion is reduced blood flow, which is found in some people with autism and also in people with some types of dementia  

Having reread my old post I would recommend it to those who are looking into the treatment of brain damage caused by ischemia. 

Brain Hypoperfusion in Autism & Cocoa

While much in neuroscience is extremely complicated, there are some pretty basic things to consider that are not. Adequate blood supply is one of the basic issues and is something that can be improved.

You can increase blood flow by reducing vascular resistance, which means reducing the work the heart has to do to circulate blood around the body. As you reduce this resistance, blood pressure will fall, but that does not mean the flow rate of blood has reduced, it just means it is circulating more freely.

You can measure cerebral blood flow and this is how researchers know that it can be abnormal in autism.

As I noted in the old post above, HBOT is one therapy proposed by some. Using an MRI you could establish with certainty if HBOT was effective in any particular individual, in regard to increasing cerebral blood flow.

I think there will be many ways to improve perfusion in an affected individual. Without a particular type of MRI you cannot really know for sure if your case of autism is one of these.

The dementia research pointed me towards cocoa flavanols, which seem to affect nitric oxide (NO), but do not directly produce it.

Nitric oxide (NO) is very important in the body and one of its roles is vasodilation (widening of blood vessels).

Some people believe that nootropic drugs work by vasodilation, i.e. more blood flow increases cognitive function.  I think that this is one of many possible ways to improve cognition, which will work in some people, but not others. 

To understand Nitric oxide (NO) you have to go a little deeper and look at eNOS (endothelial nitric oxide synthase), iNOS (inducible NO synthase) and nNOS (neuronal NO synthase). Nitric oxide can be very good for you, but it can also be very bad for you.  The short version is that Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia.,

For a thorough explanation here is a highly cited paper:-

Nitric oxide synthases: regulation and function

Endothelial NOS (eNOS, NOS III) is mostly expressed in endothelial cells. It keeps blood vessels dilated, controls blood pressure, and has numerous other vasoprotective and anti-atherosclerotic effects. Many cardiovascular risk factors lead to oxidative stress, eNOS uncoupling, and endothelial dysfunction in the vasculature. Pharmacologically, vascular oxidative stress can be reduced and eNOS functionality restored with renin- and angiotensin-converting enzyme-inhibitors, with angiotensin receptor blockers, and with statins. 

Statins are already in my Polypill. Telmisartan seemed to be the most likely ACE inhibitor or ARB (angiotensin receptor blocker) to help some autism, when I reviewed them in a previous post. Telmisartan produced more singing, as does Agmatine (see below).

Now look at how NO is produced by eNOS:-

           https://en.wikipedia.org/wiki/Endothelial_NOS 

“In the vascular endothelium, NO is synthesized by eNOS from L-arginine and molecular oxygen, which binds to the heme group of eNOS, is reduced and finally incorporated into L- arginine to form NO and L-citrulline. The binding of the cofactor BH4 is essential for eNOS to efficiently generate NO. In the absence of this cofactor, eNOS shifts from a dimeric to a monomeric form, thus becoming uncoupled. In this conformation, instead of synthesizing NO, eNOS produces superoxide anion, a highly reactive free radical with deleterious consequences to the cardiovascular system.^”

BH4 (Tetrahydrobiopterin/Kuvan) is one of substances that comes up in autism research from time to time.  You would not want to be deficient in BH4 and if you have autism and BH4 deficiency you have a very obvious therapy.   

Biomarin’s Kuvan autism treatment faces reservations on the part of experts 

A good article, surprisingly from the UK Financial Times, which they ask not to be cut and paste, so I have not. Take a look.

If Kuvan lights up the brain, as Dr Frye suggested in the above FT article, I wonder what else can, in those people.  L-arginine might help, or perhaps its metabolite Agmatine, as used by our reader Tyler.

If you read the quite complicated paper below you will see that, in rats at least, Agmatine increases eNOS, while reducing  iNOS. 

You compare EC6 (experimental control after 6 hours) with Agm6 (Agmatine after 6 hours) and then EC24 with Agm24. 

Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain

Effects of eNOS and iNOS expression by agmatine treatment following transient global ischemia in rat hippocampus. Representative expressional levels of eNOS (A) and iNOS (C) at 6 h after agmatine treatment (100 mg/kg, i.p), and densitometric data (B, D). Data represent means±SD for n=5/NC, n=3/EC and Agm group per each time point. ^*

Cost

BH4/Kuvan/Sapropterin is rather expensive, but people do use it off-label in autism.  It is the only FDA-approved medication for Phenylketonuria (PKU) to reduce blood Phe levels in patients with hyperphenylalaninemia (HPA) due to tetrahydrobiopterin (BH4-) responsive PKU.

http://www.biomarin.com/products/kuvan

PKU is one of those rare inborn errors of metabolism that lead to intellectual disability/MR and, not surprisingly, also autism. It is included in my Treatable ID tab at the top of every page.  The link will take you here  http://www.treatable-id.org/page36/Phenylketonuria.html

Agmatine is cheap and does have an almost immediate positive effect in some people with autism.

Do people who respond to BH4 respond to Agmatine and vice versa?

Agmatine does have many other modes of action, other than increasing eNOS and reducing iNOS.

I have been experimenting with Agmatine, and while Dr Frye suggests Kuvan can “light up the brain”, my impression of Agmatine brings the Energizer(US)/Duracell (Europe) Bunny to mind.

A daily dose of Agmatine is like having better battery in your toy bunny, at least in my house.  It is also associated with more singing.

Judging from Tyler’s comments perhaps he is seeing the same magnitude of effects that Dr Frye attributes to Kuvan.   

Garlic in Autism – Miscreant Microglia? ACE inhibition? or even Nitric Oxide?

Many people avoid garlic because it gives you bad breath, but if you eat enough of it, it can be a potent drug.

There is a substantial amount of research about garlic and general health - it is consistently positive. However, there is an odd resistance to tell people about it.  A good example is this quote from the website of the UK’s National Health Service.

“Studies using high concentrations of garlic extracts have been associated with improved blood circulation, healthier cholesterol levels and lower blood pressure, all of which reduce the risk of cardiovascular disease. However, current evidence does not support the use of garlic supplements to improve health.”

Which sounds like “garlic is really good for you, but don’t eat it”.

Garlic has numerous different modes of action that have a potential health benefit, the best known relate to your heart and circulation, but there are others. 

Garlic and Neurological Conditions with Activated Microglia

There is recent research showing positive effects on the activated microglia.  Activated microglia, the brain’s immune cells, is a feature of autism and other diseases, like Alzheimer’s.

Some people try and treat activated microglia in autism using therapies like:-

·        Minocycline

·        Ibudilast

Some researchers use garlic to try to minimize the damage caused by activated microglia.

They tend to use capsules that contain aged garlic.  It is important not to cook it and there is a difference between fresh garlic, aged garlic and steamed garlic. 

Table 1. Principal Organosulfur Compounds in Commercial Garlic Preparations
Product	Principal Organosulfur Compounds	Delivers allicin-derived compounds?
Fresh garlic cloves	Cysteine sulfoxides (Alliin) γ-glutamylcysteines	Yes, when chopped, crushed, or chewed raw. Minimal, when garlic cloves are cooked before crushing or chopping.
Powdered garlic (tablets)	Cysteine sulfoxides (Alliin) γ-glutamylcysteines	Varies greatly among commercial products. Enteric-coated tablets that pass the USP allicin release test are likely to provide the most.
Steam distilled garlic oil (capsules)	Diallyl disulfide Diallyl trisulfide Allyl methyl trisulfide	Yes
Garlic oil macerate (capsules)	Vinyldithiins Ajoene Diallyl trisulfide	Yes
Aged garlic extract™ (tablets or capsules)	S-Allylcysteine S-Allylmercaptocysteine S-1-Propenylcysteine	Minimal

  

Source: -http://lpi.oregonstate.edu/mic/food-beverages/garlic

  

Benefits Of Garlic Extract May Protect Brain From Inflammation, Diseases Like Alzheimer's

Now, a new study finds that one of these compounds, called FruArg, may protect the brain from age-related disease like dementia and Alzheimer’s.

As a carbohydrate derivative of garlic, there’s a relatively high concentration of FruArg in aged garlic extract (AGE), the authors wrote — AGE is typically sold as supplements. Looking at isolated FruArg’s impact on brain cells, researchers from the University of Missouri found it could protect brain cells from an overexcited immune response caused by environmental factors like pollution and smoking, as well as normal aging, brain injuries, and drinking lots of alcohol.

“Microglia are immune cells in the brain and spinal cord that are the first and main line of defense in the central nervous system,” said lead author Zezong Gu, an associate professor of pathology and anatomical sciences at the university’s School of Medicine. “Unlike other mature brain cells that seldom regenerate themselves, microglial cells respond to inflammation and environmental stresses by multiplying. By massing themselves and migrating toward an injury site, they are able to respond to inflammation and protect other brain cells from destruction.”

But microglia also tread a line between benefiting the body and harming it, protecting only to an extent. A byproduct of their function is nitric oxide, a free radical. And when a lot of microglia are produced, so are nitric oxide molecules, which can lead to oxidative stress and inflammation within the brain and nervous system. As we’ve all heard before, however, antioxidants fight oxidative stress, and in this case, that antioxidant compound is FruArg. 

For their study, Gu and his colleagues applied stress to a cell model of microglial cells and then added FruArg to them once nitric oxide concentrations rose. They found the microglial cells “adapted to the stress by reducing the amount of nitric oxide they produced.” What’s more, FruArg also promoted the production of antioxidants, which then went on to protect and heal other brain cells. “This helps us understand how garlic benefits the brain by making it more resilient to the stress and inflammation associated with neurological diseases and aging,” Gu said. 

Full study:- 

Proteomic analysis of the effects of aged garlic extract and its FruArg component on lipopolysaccharide-induced neuroinflammatory response in microglial cells.

Collectively, these results suggest that AGE and FruArg attenuate neuroinflammatory responses and promote resilience in LPS-activated BV-2 cells by suppressing NO production and by regulating expression of multiple protein targets associated with oxidative stress. 

Effects of aged garlic (AGE) extract and FruArg on gene expression and signaling pathways in lipopolysaccharide-activated microglial cells 

These effects could be modulated by treatment with both AGE and FruArg. These findings suggests that AGE and FruArg are capable of alleviating oxidative stress and neuroinflammatory responses stimulated by LPS in BV-2 cells.

  

Evaluating the Anti-Neuroinflammatory Capacity of Raw and Steamed Garlic as Well as Five Organosulfur Compounds

Abstract

: The anti-neuroinflammatory capacities of raw and steamed garlic extracts as well as five organosulfur compounds (OSCs) were examined in lipopolysaccharide (LPS)-stimulated BV2 microglia. According to those results, steaming pretreatment blocked the formation of alliinase-catalyzed OSCs such as allicin and diallyl trisulfide (DATS) in crushed garlic. Raw garlic, but not steamed garlic, dose-dependently attenuated the production of LPS-induced nitric oxide (NO), interleukin-1β (IL-1β), tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (MCP-1). DATS and diallyl disulfide at 200 and 400 μM, respectively, displayed significant anti-neuroinflammatory activity. Meanwhile, even at 1 mM, diallyl sulfide, S-allyl cysteine and alliin did not display such activity. Inhibition of nuclear factor-κB activation was the mechanism underlying this protective effect of raw garlic and DATS. Analysis results indicated that the anti-neuroinflammatory capacity of raw garlic is due to the alliin-derived OSCs. Importantly, DATS is a highly promising therapeutic candidate for treating inflammation-related neurodegenerative diseases.

As expected, raw garlic extract inhibited NO, proinflammatory cytokine, and chemokine production by through suppression of NF-κB activation in LPS-activated BV2 microglia; it also had a potent anti-neuroinflammatory capacity. Additionally, steaming pretreatment abolished both the anti-neuroinflammatory capacity and alliin-derived OSCs formation of garlic simultaneously. In sum, this study demonstrates that alliinase catalysis and chemical transformation are essential for the formation of active OSCs, which are responsible for the anti-neuroinflammatory capacity of garlic. Based on above, it is suggested that consumers to crush or cut raw garlic before cooking in order to obtain more health benefits of garlic. As one of the most potent anti-neuroinflammatory components of garlic, DATS is highly promising for use as a dietary agent to prevent inflammation-related neurodegenerative disease. 

Garlic as an ACE inhibitor 

We saw in a recent post how too much angiotensin II is likely a problem in schizophrenia and some autism.  The biomarker of those affected would be high levels of IL-17a. 

Targeting Angiotensin in Schizophrenia and Some Autism

There are numerous references in the literature to garlic being an ACE inhibitor, which will reduce the level of angiotensin II and hence IL-17 and IL-17a. 

Garlic extract reduces serum angiotensin converting enzyme (ACE) activity in nondiabetic and streptozotocin-diabetic rats. 

Although garlic extract administration had no significant effect on serum glucose, it significantly strongly decreased the serum ACE activity. ACE activity was higher in diabetic than nondiabetic rats, but in diabetic animals treated with garlic extract, the elevation of ACE activity did not occur. These results suggest that garlic extract might have value as ACE inhibitor to prevent some vascular complications of diabetes mellitus.

So perhaps some people with autism, who respond to garlic are actually not feeling the microglia effect, but actually the angiotensin II reducing effect. 

Potent activation of nitric oxide synthase by garlic: a basis for its therapeutic applications. 

Activation of calcium-dependent nitric oxide synthase and the subsequent production of nitric oxide is probably the most novel mechanism yet claimed by which garlic can exert its therapeutic properties.

   

Conclusion  

Garlic has numerous health benefits and different types of processing lead to very different chemical compositions.  So it does depend how you take your garlic.

Does any type of garlic provide a benefit in any type of autism? 

For one reader fresh garlic is effective in treating autism, whereas aged garlic is not; this is not what she expected. This would of course suggest something about its mode of action. 

Perhaps some people are actually benefiting from a reduction in angiotensin II.  Or maybe it is production of nitric oxide?

There are actually other natural ACE inhibitors that you might be using by accident.

People trying to make tasty drinkable sulforaphane, using the Australian mixture of broccoli and pomegranate powders, are actually also making an ACE inhibitor.  

Effect of pomegranate juice on Angiotensin II-induced hypertension in diabetic wistarrats

The results suggest that the PJ extract could prevent the development of high blood pressure induced by Ang II in diabetic rats probably by combating the oxidative stress induced by diabetes and Ang II and by inhibiting ACE activity.

Pharmacological review on Natural ACE inhibitors 

All we can say is some people with autism respond to specific types of garlic, but nobody can be sure what the mode of action is; there are several possible credible explanations.