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Showing posts with label Complex I. Show all posts
Showing posts with label Complex I. Show all posts

Wednesday, 3 October 2018

Ketones and Autism Part 6 - Capric Acid (C10) for Mitochondrial Disease, in Particular Complex 1, plus more on Metformin



Capric Acid (C10) is so named because it smells like a goat (Goat in Latin = Caper)
Photographer: Armin Kübelbeck, CC-BY-SA, Wikimedia Commons

Rather than Goaty acid, C10 is called Capric acid, or indeed Decanoic acid (after its 10 carbon atoms). Today’s post is indirectly again about ketones, because if you eat a Ketogenic Diet (KD) you are likely to consume a fair amount of Capric acid (C10).
I have written a lot in this blog about mitochondria, even though I do not think my son has mitochondrial dysfunction. Clearly many people with autism do have a lack of one or more of the critical mitochondrial enzyme complexes that allow glucose to be converted to ATP (usable energy), by the clever process OXPHOS (Oxidative phosphorylation).

The “rate limiting” enzyme is usually Complex 1, meaning that is the one it is most important not to be short of.
Another favourite, but obscure, subject of this blog is PPAR gamma.

Peroxisome proliferator-activated receptors (PPARs) are a group of proteins that function as transcription factors regulating the expression of certain genes. Transcription factors are particularly important because they trigger numerous effects.
PPAR gamma plays a key role in fat storage and glucose metabolism, but has other functions. 

Activation of PPAR-gamma by Capric acid (C10) has been shown to increase the number of mitochondria, increase the mitochondrial enzyme citrate synthase, increase complex I activity in mitochondria, and increase activity of the antioxidant enzyme catalase. 
So, if you have autism and impaired mitochondrial function, C10 may well give a benefit because it can increase the peak power available to your brain.


The Ketogenic diet (KD) is an effective treatment with regards to treating pharmaco-resistant epilepsy. However, there are difficulties around compliance and tolerability. Consequently, there is a need for refined/simpler formulations that could replicate the efficacy of the KD. One of the proposed hypotheses is that the KD increases cellular mitochondrial content which results in elevation of the seizure threshold. Here, we have focussed on the medium-chain triglyceride form of the diet and the observation that plasma octanoic acid (C8) and decanoic acid (C10) levels are elevated in patients on the medium-chain triglyceride KD. Using a neuronal cell line (SH-SY5Y), we demonstrated that 250-μM C10, but not C8, caused, over a 6-day period, a marked increase in the mitochondrial enzyme, citrate synthase along with complex I activity and catalase activity. Increased mitochondrial number was also indicated by electron microscopy. C10 is a reported peroxisome proliferator activator receptor γ agonist, and the use of a peroxisome proliferator activator receptor γ antagonist was shown to prevent the C10-mediated increase in mitochondrial content and catalase. C10 may mimic the mitochondrial proliferation associated with the KD and raises the possibility that formulations based on this fatty acid could replace a more complex diet. We propose that decanoic acid (C10) results in increased mitochondrial number. Our data suggest that this may occur via the activation of the PPARγ receptor and its target genes involved in mitochondrial biogenesis. This finding could be of significant benefit to epilepsy patients who are currently on a strict ketogenic diet. Evidence that C10 on its own can modulate mitochondrial number raises the possibility that a simplified and less stringent C10-based diet could be developed.

Capric Acid (C10) as a PPARγ agonist

As shown in the above study the mechanism by which C10 benefits the mitochondria is via PPARγ agonism.

Here is another study confirming that C10 is indeed a PPARγ agonist.


Background: Mechanism of action of medium chain fatty acid remains unknown.

Results: Our results show that decanoic acid (C10) binds and activates PPARγ.

Conclusion: Decanoic acid acts as a modulator of PPARγ and reduces blood glucose levels with no weight gain.

Significance: This study could lead to design of better type 2 diabetes drugs.


Other PPARγ agonists
PPARγ agonists have been covered previously in this blog and we know that glitazones, a class of drugs for diabetes, do improve some types of autism. Glitazones are PPARγ agonists.

Metformin, a very widely used drug for type 2 diabetes, works differently to Glitazones, but I did suggest a while back it should help some types of autism. Last year it was indeed found to be beneficial in Fragile X.


 "Basically, it's something like a wonder drug," Sonenberg said.
The study suggests that metformin might also be used to treat other autism spectrum disorders, said Ilse Gantois, a research associate in Sonenberg's lab at McGill.
"We mostly looked at the autistic form of behaviour in the Fragile X mouse model," explained Gantois, who is co-lead author with McGill researchers Arkady Khoutorsky and Jelena Popic. "We want to start testing other mouse models to see if the drug could also have benefits for other types of autism."

Metformin is very cheap and has been used in humans for 60 years. It is another example of re-purposing a drug from Grandpa’s medicine cabinet to treat Grandson’s autism. 

Metformin has been trialled to combat obesity in idiopathic autism caused by antipsychotics. It did help with weight gain, but no comments were made about behavioural improvements, but then those studied were on antipsychotic drugs, which might mask such effects. 
Glitazone-type drugs appear more problematic than Metformin.

There are natural PPAR gamma agonists and they are often used to lower cholesterol, lower blood sugar and improve insulin sensitivity.
Sytrinol, a product containing flavanols tangeretin and nobiletin does indeed have a positive effect on some people’s autism, but for most people (but not all) the effect is lost after a few days.

Our doctor reader Maja, did suggest combining it with a PPARα agonist to see if the effect might be maintained.
This combination has indeed been researched for type 2 diabetes.               

The effect of dual PPAR alpha/gamma stimulation with combination of rosiglitazone and fenofibrate on metabolic parameters in type 2 diabetic patients.


There actually is another natural substance that is an agonist of both PPARγ and PPARα, Berberine, the alkaloid long used in Chinese medicine.
In the research it is suggested that BRB localizes in mitochondria, inhibits respiratory electron chain and activates AMPK”, which is not what you would want. But this may not be correct.

People who like supplements might want to follow up on Berberine.
Berberine is used by many people with diabetes and a few with autism, for all kinds of reasons, from mercury to GI problems.

Berberine is a potent agonist of peroxisome proliferator activated receptor alpha.


Although berberine has hypolipidemic effects with a high affinity to nuclear proteins, the underlying molecular mechanism for this effect remains unclear. Here, we determine whether berberine is an agonist of peroxisome proliferator-activated receptor alpha (PPARalpha), with a lipid-lowering effect. The cell-based reporter gene analysis showed that berberine selectively activates PPARalpha (EC50 =0.58 mM, Emax =102.4). The radioligand binding assay shows that berberine binds directly to the ligand-binding domain of PPARalpha (Ki=0.73 mM) with similar affinity to fenofibrate. The mRNA and protein levels of CPT-Ialpha gene from HepG2 cells and hyperlipidemic rat liver are remarkably up-regulated by berberine, and this effect can be blocked by MK886, a non-competitive antagonist of PPARalpha. A comparison assay in which berberine and fenofibrate were used to treat hyperlipidaemic rats for three months shows that these drugs produce similar lipid-lowering effects, except that berberine increases high-density lipoprotein cholesterol more effectively than fenofibrate. These findings provide the first evidence that berberine is a potent agonist of PPARalpha and seems to be superior to fenofibrate for treating hyperlipidemia.


                                                                                                                                     

Sources of Capric Acid (C10)
Goat milk is a good source of capric acid.
Capric acid is 8-10% of coconut oil and 4% of palm kernel oil

Capric acid is a large component (about 40%) of the less expensive MCT oil supplements.


1.2. Fatty acid composition in goat milk fat Average goat milk fat differs in contents of its fatty acids significantly from average cow milk fat, being much higher in butyric (C4:0), caproic (C6:0), caprylic (C8:0), capric (C10:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), linoleic (C18:2), but lower in stearic (C18:0), and oleic acid (C18:1) (Table 1). Three of the medium chain fatty acids (caproic, caprylic, and capric) have actually been named after goats, due to their predominance in goat milk. They contribute to 15% of the total fatty acid content in goat milk in comparison to 5% in cow milk (Haenlein, 1993). The presence of relatively high levels of medium chain fatty acids (C6:0 to C10:0) in goat milk fat could be responsible for its inferior flavour (Skjevdal, 1979). 

             
Conclusion
If someone responds well to coconut oil or cheaper MCT oil the reason may have more to do with PPAR gamma and improved mitochondrial function than anything to do with ketones and what they do.
Cheaper MCT oils are mainly a mixture of C8 and C10. To maximize the production of the ketone BHB you really want just C8, but if what you really need is a PPAR gamma agonist, to perk up your mitochondria, it is the C10 you need.
You may indeed benefit from both ketones and agonizing PPAR gamma, in which case you either follow the Ketogenic Diet, or supplement BHB, C8 and C10.
I think this explains why some people with autism reportedly respond well to teaspoon-sized doses of cheaper MCT oil or small amounts of coconut oil.
If you have Complex 1 mitochondrial dysfunction then a dose of Capric acid (C10) is likely to help.
Berberine may, or may not be, as effective as C10. I doubt we will ever know. I think C10 is the better option. 
I wonder when the Canadian researchers will publish their results showing whether Metformin is beneficial beyond Fragile X syndrome. They do not really know why it helps, but that is a repeating theme in medicine.  It is a cheap safe drug, so it would be a pity to waste time finding out if it could be repurposed for some autism.



Friday, 1 June 2018

Autism, Power Outages and the Starving Brain?



There are certain Critical Periods in the development of the human brain and these are the most vulnerable times to any genetic or environmental insult.  Critical Periods (CPs) will be the subject of post appearing shortly.


Another power outage waiting to happen

 Have you wondered why autism secondary to mitochondrial disease (regressive autism) almost always seem to occur before five years of age, and usually much earlier?  Why does it not happen later? Why is it's onset often preceded by a viral infection?
I think you can consider much of this in terms of the brain running out of energy. Humans have evolved to require a huge amount of energy to power their developing brains, a massive 40% of the body’s energy is required by the brain in early childhood.  If your overload a power grid it will end in a blackout.
We know many people with autism have a tendency towards mitochondrial dysfunction, they lack some key enzyme complexes. This means that the process of OXPHOS (Oxidative phosphorylation), by which the body converts glucose to usable energy (ATP), is partially disabled. 

We saw in earlier posts how the supply of glucose and oxygen to the brain can be impaired in autism because there is unstable blood flow.


It is just like in your house, all your electrical appliances might mean you need a 25KW supply, because you do not use them all at the same time. Just to be on the safe side you might have a 40KW limit. What if the power company will only give you a 20 KW connection? If you turn on the clothes drier, the oven, the air conditioning and some other things all of a sudden you blow the main fuse and perhaps damage the hard drive of your old computer.
So, in the power-hungry brain of a three-year-old, you add a viral infection and all of a sudden you exceed the available power supply from the mitochondria, that have soldered on for 3years with impaired supply of complex 1 and imperfect cerebral blood flow. By the sixth year of life, the peak power requirement from the brain would have fallen to within the safe limit of the mitochondria and its impaired supply of complex 1.  Instead of blowing the fuse, which is easy to reset, you have blown some neuronal circuitry, which is not so easy to repair.    

Too Many Synapses?
We know that it is the synapses in the brain that are the big energy users and we also know that in most autism there are too many synapses. So, in that group of autism there is an even bigger potential energy demand.



Note that in Alzheimer’s type dementia (AD in the above chart) you see a severe loss of synapses/spines as atrophy takes place. This occurs at the same time as a loss of insulin sensitivity occurs (type 3 diabetes). Perhaps the AD brain is also starved of energy, it does seem to respond to ketosis (ketones replacing glucose as the fuel) and it responds to Agmatine (increasing blood flow via eNOS).
We also know that adolescent synaptic pruning is dysfunctional in autism and we even know why. Interestingly by modifying GABAA function with bumetanide we may indeed allow the brain to eliminate more synapses (a good thing), so possibly an unexpected benefit from Ben Ari’s original idea.

"Working with a mouse model we have shown that, at puberty, there is an increase in inhibitory GABA receptors, which are targets for brain chemicals that quiet down nerve cells. We now report that these GABA receptors trigger synaptic pruning at puberty in the mouse hippocampus, a brain area involved in learning and memory." The report, published by eLife, "Synaptic pruning in the female hippocampus is triggered at puberty by extrasynaptic GABAA receptors on dendritic spines."            
These findings may suggest new treatments targeting GABA receptors for "normalizing" synaptic pruning in diseases such as autism and schizophrenia, where synaptic pruning is abnormal. Research has suggested that children with autism may have an over-abundance of synapses in some parts of the brain.

Synaptic pruning in the female hippocampus is triggered at puberty by extrasynaptic GABAA receptors on dendritic spines

Adolescent synaptic pruning is thought to enable optimal cognition because it is disrupted in certain neuropathologies, yet the initiator of this process is unknown. One factor not yet considered is the α4βδ GABAA receptor (GABAR), an extrasynaptic inhibitory receptor which first emerges on dendritic spines at puberty in female mice. Here we show that α4βδ GABARs trigger adolescent pruning. Spine density of CA1 hippocampal pyramidal cells decreased by half post-pubertally in female wild-type but not α4 KO mice. This effect was associated with decreased expression of kalirin-7 (Kal7), a spine protein which controls actin cytoskeleton remodeling. Kal7 decreased at puberty as a result of reduced NMDAR activation due to α4βδ-mediated inhibition. In the absence of this inhibition, Kal7 expression was unchanged at puberty. In the unpruned condition, spatial re-learning was impaired. These data suggest that pubertal pruning requires α4βδ GABARs. In their absence, pruning is prevented and cognition is not optimal.


Strange Patterns of Growth
Longitudinal studies are when researchers collect the same data over long period of years. Most autism research is just based on a single snapshot in time.
One observation of mine is that some people with strictly defined autism (SDA) are born at the 90+ percentile for height, but then fall back to something like the 20 percentile. Body growth has dramatically slowed. Was this because energy has been diverted to the overgrowing brain? 
A five-year old’s brain is an energy monster. It uses twice as much glucose (the energy that fuels the brain) as that of a full-grown adult, a new study led by Northwestern University anthropologists has found.
It was previously believed that the brain’s resource burden on the body was largest at birth, when the size of the brain relative to the body is greatest. The researchers found instead that the brain maxes out its glucose use at age 5. At age 4 the brain consumes glucose at a rate comparable to 66 percent of the body’s resting metabolic rate (or more than 40 percent of the body’s total energy expenditure). 

“The mid-childhood peak in brain costs has to do with the fact that synapses, connections in the brain, max out at this age, when we learn so many of the things we need to know to be successful humans,” Kuzawa said.

“At its peak in childhood, the brain burns through two-thirds of the calories the entire body uses at rest, much more than other primate species,” said William Leonard, co-author of the study. “To compensate for these heavy energy demands of our big brains, children grow more slowly and are less physically active during this age range. Our findings strongly suggest that humans evolved to grow slowly during this time in order to free up fuel for our expensive, busy childhood brains.” 

Full paper: -


The high energetic costs of human brain development have been hypothesized to explain distinctive human traits, including exceptionally slow and protracted preadult growth. Although widely assumed to constrain life-history evolution, the metabolic requirements of the growing human brain are unknown. We combined previously collected PET and MRI data to calculate the human brain’s glucose use from birth to adulthood, which we compare with body growth rate. We evaluate the strength of brain–body metabolic trade-offs using the ratios of brain glucose uptake to the body’s resting metabolic rate (RMR) and daily energy requirements (DER) expressed in glucose-gram equivalents (glucosermr% and glucoseder%). We find that glucosermr% and glucoseder% do not peak at birth (52.5% and 59.8% of RMR, or 35.4% and 38.7% of DER, for males and females, respectively), when relative brain size is largest, but rather in childhood (66.3% and 65.0% of RMR and 43.3% and 43.8% of DER). Body-weight growth (dw/dt) and both glucosermr% and glucoseder% are strongly, inversely related: soon after birth, increases in brain glucose demand are accompanied by proportionate decreases in dw/dt. Ages of peak brain glucose demand and lowest dw/dt co-occur and subsequent developmental declines in brain metabolism are matched by proportionate increases in dw/dt until puberty. The finding that human brain glucose demands peak during childhood, and evidence that brain metabolism and body growth rate covary inversely across development, support the hypothesis that the high costs of human brain development require compensatory slowing of body growth rate. 

To quantify the metabolic costs of the human brain, in this study we used a unique, previously collected age series of PET measures of brain glucose uptake spanning birth to adulthood (32), along with existing MRI volumetric data (36), to calculate the brain’s total glucose use from birth to adulthood, which we compare with body growth rate. We estimate total brain glucose uptake by age (inclusive of all oxidative and nonoxidative functions), which we compare with two measures of whole-body energy expenditure: RMR, reflecting maintenance functions only, and daily energy requirements (DER), reflecting the combination of maintenance, activity, and growth. We hypothesized that ages of peak substrate competition (i.e., competition for glucose) between brain and body would be aligned developmentally with the age of slowest childhood body growth, and more generally that growth rate and brain glucose use would covary inversely during development, as is predicted by the concept of a trade-off between brain metabolism and body growth in human life-history evolution. 

Daily glucose use by the brain peaks at 5.2 y of age at 167.0 g/d and 146.1 g/d in males and females, respectively. These values represent 1.88- and 1.82-times the daily glucose use of the brain in adulthood (Fig. 1 A and B and SI Appendix, Fig. S2), despite the fact that body size is more than three-times as large in the adult.




Glucose use of the human brain by age. (A) Grams per day in males. (B) Grams per day in females; dashed horizontal line is adult value (A and B). (C) Glucosermr% (solid line) and glucoseder% (dashed line) in males. (D) Glucosermr% (solid line) and glucoseder% (dashed line) in females.

The most relevant data is the line highlighted in yellow below, showing brain consumption of glucose peaks at 40% (of total body consumption) around 5 years old and drops to 20% in adulthood.

Our findings agree with past estimates indicating that the brain dominates the body’s metabolism during early life (31). However, our PET-based calculations reveal that the magnitude of brain glucose uptake, both in absolute terms and relative to the body’s metabolic budget, does not peak at birth but rather in childhood, when the glucose used by the brain comprises the equivalent of 66% of the body’s RMR, and roughly 43% of total expenditure. These findings are in broad agreement with past clinical work showing that the body’s mass-specific glucose production rates are highest in childhood, and tightly linked with the brain’s metabolic needs (40). Whereas past attempts to quantify the contribution of the brain to the body’s metabolic expenditure suggested that the brain accounted for a continuously decreasing fraction of RMR as the brain-to-body weight ratio declined with age (25, 31), we find a more complex pattern of substrate trade-off. Both glucosermr% and glucoseder% decline in the first half-year as a fast but decelerating pace of body growth established in utero initially outpaces postnatal increases in brain metabolism. Beginning around 6 mo, increases in relative glucose use are matched by proportionate decreases in weight growth, whereas ages of declining brain glucose uptake in late childhood and early adolescence are accompanied by proportionate increases in weight growth. The relationships that we document between age changes in brain glucose demands and body-weight growth rate are particularly striking in males, who maintain these inverse linear trends despite experiencing threefold changes in brain glucose demand and body growth rate between 6 mo and 13 y of age. In females, an earlier onset of pubertal weight gain leads to earlier deviations from similar linear inverse relationships.
                                     

What the researchers then did was to see how the growth rate of the brain is correlated to the growth rate of the body. In effect that what they found was that the growth of the body has to slow down to allow the energy hungry brain to develop.  One the brain has passed its peak energy requirement at about 5 years old, body growth can then gradually accelerate. 
The brain is the red line, the body is blue. The chart on the left is males and the one on the right is females. 
So, we might suspect that in 2 to 4-year olds who seem not to be growing as fast as we might expect, the reason is that their brain is over-growing, a key feature of classic autism.

Glucoseder% and body-weight growth rate. Glucoseder% and weight velocities plotted as SD scores to allow unitless comparison. (A) Glucoseder% (red dots) and dw/dt (blue dots) by age in males. (B) Glucoseder% (red dots) and dw/dt (blue dots) by age in females


Brain Overgrowth in Autism
As has been previous commented on in this blog, Eric Courchesne has pretty much figured out what goes wrong in the growth trajectory of the autistic brain; that was almost 15 years ago.

Brain development in autism: early overgrowth followed by premature arrest of growth.


Author information


Abstract


Due to the relatively late age of clinical diagnosis of autism, the early brain pathology of children with autism has remained largely unstudied. The increased use of retrospective measures such as head circumference, along with a surge of MRI studies of toddlers with autism, have opened a whole new area of research and discovery. Recent studies have now shown that abnormal brain overgrowth occurs during the first 2 years of life in children with autism. By 2-4 years of age, the most deviant overgrowth is in cerebral, cerebellar, and limbic structures that underlie higher-order cognitive, social, emotional, and language functions. Excessive growth is followed by abnormally slow or arrested growth. Deviant brain growth in autism occurs at the very time when the formation of cerebral circuitry is at its most exuberant and vulnerable stage, and it may signal disruption of this process of circuit formation. The resulting aberrant connectivity and dysfunction may lead to the development of autistic behaviors. To discover the causes, neural substrates, early-warning signs and effective treatments of autism, future research should focus on elucidating the neurobiological defects that underlie brain growth abnormalities in autism that appear during these critical first years of life.


Research from 2017: -





Conclusion
A record of children’s height and weight and even head circumference is usually collected by their doctor. In an earlier post I did ask why they bother if nobody is checking this data. If a child falls from the 90th percentile in height to the 20th, something clearly is going on.
When I discussed this with a pediatric endocrinologist a few years ago, we then measured bone-age and IGF-1. If you have low IGF-1 and retarded bone age you might opt for some kind of growth hormone therapy.
In what is broadly defined as autism, I think we have some distinctly different things possibly happening: -

Group AMD
Energy conversion in the brain is less efficient than it should be due to a combination of impaired vascular function and impaired mitochondrial enzyme complex production. No symptoms are apparent and developmental milestones are achieved.  As the brain creates more synapses it energy requirement grows until the day when the body has some external insult like a viral infection, and the required power is not available, triggering a “power outage” which appears as the regression into autism. In biological terms there has been death of neurons and demyelination.

Group Sliding Down the Percentiles 
This group looks like a sub-set of classic autism. The brain grows too rapidly in the first two years after birth and this causes the expected slowing of body growth to occur much earlier than in typical children. This manifests itself in the child tumbling down the percentiles for height and weight.
The brain then stops growing prematurely, reducing energy consumption and allowing body growth to accelerate and the child slowly rises back up the height/weight percentiles.

Perhaps all those excessive synapses that were not pruned correctly are wasting glucose and so delay the growth of the rest of the body?   
In the sliding down the growth percentiles group, does this overgrowing brain ever exceed maximum available power? Maybe it just grows too fast and so mal-develops, as suggested by Courchesne, or maybe it grows too fast and cannot fuel correct development?  What happens if you increase maximum available power in this group, in the way some athletes use to enhance their performance/cheat?
All I know for sure is that in Monty, aged 14 with autism, increasing eNOS (endothelial nitric oxide synthase) using agmatine seems to make him achieve much more, with the same daily glucose consumption. I wonder what would happen if Agmatine was given to very young children as soon as it was noted that they were tumbling down the height percentiles?  This is perhaps what the pediatric endocrinologists should be thinking about, rather than just whether or not to administer growth hormones/IGF-1.
If you could identify Group AMD before the “power outage” you might be able to boost maximum power production or reduce body growth slightly and hence avoid the brain ever being starved of energy. That way you would not have most regressive autism.







Tuesday, 21 February 2017

Mitochondrial Disease and Autsim




Today’s post was originally intended to look at some further methods used to enhance cognitive function. Unlike people with typical mild cognitive impairment (MCI), some people with autism exhibit highly variable cognitive function, one way this is visible is in their hand writing quality. We previously saw that in cases of PANDAS/PANS, deterioration of hand writing is also seen during acute episodes. 
One possible cause of cognitive decline is mitochondrial dysfunction.  This is a highly complex subject in its own right and so I decided to start with a post introducing mitochondrial disease and dysfunction.
  
Mitochondria

Mitochondria are tiny organelles found in almost every cell in the body. These organelles are responsible for creating 90% of cellular energy necessary to maintain life and support growth. Mitochondrial disease occurs when mitochondria in the cells fail to produce enough energy to sustain cell life. When enough cells cease to function properly organs, motor functions, and the neurological system can become impaired.
Mitochondrial disease is often misdiagnosed due to the fact many of the symptoms are synonymous with other, more common, diseases.
In more scientific terms mitochondrial disease refers to a wide ranging group of disorders resulting in defective cellular energy production due to abnormal oxidative phosphorylation (OXPHOS), which is explained a little later.

Primary Mitochondrial Disease (PMD) vs Secondary Mitochondrial Dysfunction (SMD)
I received a comment a while back from a parent who said that tests had ruled out mitochondrial disease.  It is actually a very grey area, where it is much easier to rule it in, than out. It looks like most people with autism have some mitochondrial dysfunction, albeit perhaps minor compared to those with an identified error in a critical gene, which is today relatively easy to diagnose.

Primary Mitochondrial Disease (PMD) is inborn; people with PMD gave a genetic variance that makes them vulnerable to a loss of mitochondrial function.  This loss may not begin until later in life and may increase in severity.
PMD is extremely rare in the general population, but is thought to occur in about 5% of cases of autism.
Primary mitochondrial disease (PMD) is diagnosed clinically and ideally, but not always, confirmed by a known or indisputably pathogenic mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) mutation. The PMD genes either encode oxphos proteins directly or they affect oxphos function by impacting production of the complex machinery needed to run the oxphos process.
Secondary mitochondrial dysfunction (SMD) is much more common than PMD. SMD can be caused by genes encoding neither function nor production of the oxphos proteins and accompanies many hereditary non-mitochondrial diseases. SMD may also be due to non-genetic causes such as environmental factors.
SMD has been documented in a variety of autoimmune processes including multiple sclerosis and lupus.
Aging contributes to oxidative stress in virtually all organs and tissues in the body and increases the risk for SMD.
Altered mitochondrial fusion/fission dynamics have been found to be a recurring theme in neurodegeneration. There is evidence of mitochondrial dysfunction in neurodegenerative diseases such as Alzheimer's and Parkinson's.
A significant number of metabolic disorders include SMD as a part of their phenotypes.
Abnormal biomarkers of mitochondrial function are very common in autism.  Depending on whose data you consider, you can say that SMD is present in a substantial minority or even a majority of cases.
Ideally you would use genetic testing to try to distinguish between PMD and SMD. This is important, since their treatments and prognoses can be quite different. However, even in the absence of the ability to distinguish between PMD and SMD, treating SMD with standard treatments for PMD can be effective.

Diagnosis of PMD, SMD and specific subtypes
Some researchers/clinicians make the issue of diagnosis sound very clear cut, whereas others see it as a subjective diagnosis associated with some “ifs” and “maybes”.

Mitochondrial dysfunction can affect the whole body or be organ specific. You can take a muscle biopsy for analysis but not a brain biopsy.
There are a small number of well-known specialists who diagnose mitochondrial dysfunction. They all have their own favoured treatments and they do vary.  


Oxidative phosphorylation
Oxidative phosphorylation (or OXPHOS in short) is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy.  This takes place inside mitochondria.

Although oxidative phosphorylation is a vital part of metabolism, it produces reactive oxygen species such as superoxide and hydrogen peroxide, which lead to propagation of free radicals, damaging cells and contributing to disease.
The five enzymes required have simplified names: complex I, complex II, complex III, complex IV, and complex V.
In the mitochondria, converting one molecule of glucose to carbon dioxide and water produces up to 36 ATPs. This does also require the presence of oxygen, in the absence of oxygen a different, much less efficient process is followed.  This is where some sportsmen seek to cheat by increasing the amount of oxygen in their blood.
Adenosine triphosphate (ATP) is a small molecule used in cells as a coenzyme. It is often referred to as the "molecular unit of currency" of intracellular energy transfer.

ATP transports chemical energy within cells for metabolism. Most cellular functions need energy in order to be carried out: synthesis of proteins, synthesis of membranes, movement of the cell, cellular division, transport of various solutes etc. ATP is the molecule that carries energy to the place where the energy is needed.

When ATP breaks into ADP (Adenosine diphosphate) and Pi (phosphate), energy is liberated.
By analyzing the level of certain byproducts of the five major steps between glucose and ATP you can determine which of the five enzyme complexes might be deficient.
Many poisons and pesticides target one of the enzyme complexes.  Inhibition of any step in this process will halt the rest of the process. One of these poisons, 2,4-Dinitrophenol, was actually used as an anti-obesity drug in the 1930s.


Complex I to V in Autism
The clinicians who like genetic testing look for concrete evidence of Primary Mitochondrial Disease (PMD). Other clinicians look for tell-tale signs in the level of chemicals like lactate and pyruvate to make diagnosis; this might suggest that a specific enzyme complex is deficient.

So if you have a diagnosis of say complex 1 deficiency, you can then go into the detail of that step in the process.  Here is gets rather complicated because 51 different genes encode components of complex 1.  Any one of them being down regulated could impair the level of complex 1.  


The researchers obtained blood samples from each child and analyzed the metabolic pathways of mitochondria in immune cells called lymphocytes. Previous studies sampled mitochondria obtained from muscle, but the mitochondrial dysfunction sometimes is not expressed in muscle. Muscle cells can generate much of their energy through anaerobic glycolysis, which does not involve mitochondria. By contrast, lymphocytes, and to a greater extent brain neurons, rely more heavily on the aerobic respiration conducted by mitochondria.

The researchers found that mitochondria from children with autism consumed far less oxygen than mitochondria from the group of control children, a sign of lowered mitochondrial activity. For example, the oxygen consumption of one critical mitochondrial enzyme complex, NADH oxidase, in autistic children was only a third of that found in control children. 

Complex I was the site of the most common deficiency, found in 60 percent of autistic subjects, and occurred five out of six times in combination with Complex V. Other children had problems in Complexes III and IV.

Levels of pyruvate, the raw material mitochondria transform into cellular energy, also were elevated in the blood plasma of autistic children. This suggests the mitochondria of children with autism are unable to process pyruvate fast enough to keep up with the demand for energy, pointing to a novel deficiency at the level of an enzyme named pyruvate dehydrogenase.

"The various dysfunctions we measured are probably even more extreme in brain cells, which rely exclusively on mitochondria for energy," 
"Children with mitochondrial diseases may present exercise intolerance, seizures and cognitive decline, among other conditions. Some will manifest disease symptoms and some will appear as sporadic cases," said Cecilia Giulivi, the study's lead author and professor in the Department of Molecular Biosciences in the School of Veterinary Medicine at UC Davis. "Many of these characteristics are shared by children with autism."

Therapy
It looks like Dr Kelley, formerly at Johns Hopkins, has the largest following by those treating autism secondary to mitochondrial disease (AMD). Treatment includes augmentation of residual complex I activity with carnitine, thiamine, nicotinamide, and pantothenate, and protection against free radical injury with several antioxidants, including vitamin C, vitamin E, alpha-lipoic acid, and coenzyme Q10.
Dr Frye is a prolific publisher, unlike Dr Kelley, and their therapies do differ.  The table below is from one of Dr Frye’s papers.



Dr Frye likes his B vitamins. On his list are B vitamins  1,2,3,5,6,7,9 and 12


Dr Kelley is a big believer in the benefit of carnitine:


“Mutation in one or more subunits of mitochondrial complex I in AMD also is suggested by the often immediate response to carnitine, which activates latent complex I by the same NDUSF7/phosphatase-kinase system that activates pyruvate dehydrogenase.  Although immediate behavioral improvement with carnitine treatment in a child with regressive autism makes complex I deficiency the most likely cause, the similar effect of carnitine to activate latent pyruvate dehydrogenase complex recommends consideration of pyruvate dehydrogenase deficiency in the child with atypical autism and substantial postprandial lactic acidemia.”

“Supplemental carnitine enhances the conversion of acyl-CoAs to free CoA + acylcarnitines, thereby raising the intramitochondrial free CoA/acyl-CoA ratio and activating the phosphatase that reverses the inhibitory phosphorylation of NDUFS7.  Pharmacological amounts of pantothenic acid increase the synthesis of free CoA in mitochondria [22], which increases further the free-CoA/acyl-CoA ratio.  Raising the free-CoA/acyl-CoA ratio recruits more functional complex I units to compensate for the partial deficiency of complex I.  Because complex I is the rate limiting step in the mitochondrial respiratory chain for most substrates, each percentage increase in complex I activity should be followed by a substantial fraction of that percentage increase in mitochondrial ATP synthesis  

A problem with carnitine is very low bioavailability. 


Carnitine is important for cell function and survival primarily because of its involvement in the multiple equilibria between acylcarnitine and acyl-CoA esters established through the enzymatic activities of the family of carnitine acyltransferases. These have different acyl chain-length specificities and intracellular compartment distributions, and act in synchrony to regulate multiple aspects of metabolism, ranging from fuel-selection and -sensing, to the modulation of the signal transduction mechanisms involved in many homeostatic systems. This review aims to rationalise the extensive range of experimental and clinical data that have been obtained through the pharmacological use of L-carnitine and its short-chain acylesters, over the past two decades, in terms of the basic biochemical mechanisms involved in the effects of carnitine on the various cellular acyl-CoA pools in health and disease.


4.3. L-Carnitine: a conditional drug?

The potential limitation of L-carnitine-based “mitochondrial” therapy may be overcome through the attainment of supraphysiological concentrations of L-carnitine in plasma and target organs, so as to elicit the desired pharmaco-metabolic response. In target organs such as liver, heart, and skeletal muscle, the intracellular L-carnitine pool is in the high micromolar to low millimolar range, whereas in the plasma it is in the low micromolar range [124]. In addition, taking into account that physiological plasma levels of L-carnitine almost saturate the high-affinity L-carnitine transporters, relatively high L-carnitine
plasma exposures are required to significantly achieve organ Lcarnitine
increases. Under these conditions, it is possible that Lcarnitine moves into the intracellular milieu via passive diffusion and/or a low-affinity carnitine transporter [125]. However, the increase of L-carnitine plasma exposure upon L-carnitine oral administration, even when using high doses (e.g. more than 2 grams per day) [124], is quite modest, since L-carnitine has a very poor absorption and bioavailability, a very high renal clearance, and active uptake into tissues by a high-affinity transporter [124,125]. Intravenous administration of L-carnitine might overcome such a problem, particularly for acute/short-term treatment of hospitalized patients. However, this route of administration may present difficulties, particularly when kidney function is intact, because the efficient tubular reabsorption process ensures that more than 95% of L-carnitine filtered by glomeruli is retained [124,126]. Moreover, since renal tubular
reabsorption occurs via an active transporter, once the transporter
is saturated the excess of exogenous L-carnitine is readily excreted.
  
A Carnitine Analog Perhaps?
I did write a post about Meldonium/Mildronate, a drug that was made famous by the Russian tennis star Maria Sharapova.  This drug was developed in Latvia.

One of its effects is thought to be increasing the size of blood vessels and therefore improving blood flow; this increases exercise endurance.
This fact was very well known in the old Soviet Union and Meldonium was widely used by their soldiers fighting in Afghanistan.  At high altitudes there is less oxygen in the air you breathe and ultimately less in your blood and this compromises the ability of infantry soldiers.
The western world’s military have long used  acetazolamide/Diamox which makes your blood more acidic and this  fools the body into thinking it has an excess of CO2, and it excretes this imaginary excess CO2 by deeper and faster breathing, which in turn increases the amount of oxygen in the blood.   
Other than sportswomen and soldiers, Meldonium is used to treat coronary artery disease, where problems may sometimes lead to ischemia, a condition where too little blood flows to the organs in the body, especially the heart. Because this drug is thought to expand the arteries, it helps to increase the blood flow as well as increase the flow of oxygen throughout the body.
Meldonium also appears to have neuroprotective properties particularly relevant to the mitochondria.  At one point I thought this was just the Latvian researchers clutching at straws trying to push their drug as a panacea.
Rather, I think perhaps its core action may include making the mitochondria work a little better, by increasing complex 1. This might also increase stamina and it should also improve cognition in some.

Mildronate has a very similar structure to carnitine.






 Previously, we have found that mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a small molecule with charged nitrogen and oxygen atoms, protects mitochondrial metabolism that is altered by inhibitors of complex I and has neuroprotective effects in an azidothymidine-neurotoxicity mouse model


The aim of this study was to investigate: (1) whether mildronate may protect mitochondria from AZT-induced toxicity; and (2) which is the most critical target in mitochondrial processes that is responsible for mildronate's regulatory action. The results showed that mildronate protected mitochondria from AZT-induced damage predominantly at the level of complex I, mainly by reducing hydrogen peroxide generation. Significant protection of AZT-caused inhibition of uncoupled respiration, ADP to oxygen ratio, and transmembrane potential were also observed. Mildronate per se had no effect on the bioenergetics, oxidative stress, or permeability transition of rat liver mitochondria. Since mitochondrial complex I is the first enzyme of the respiratory electron transport chain and its damage is considered to be responsible for different mitochondrial diseases, we may account for mildronate's effectiveness in the prevention of pathologies associated with mitochondrial dysfunctions.




Previously we demonstrated that mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a representative of the aza-butyrobetaine class of compounds, protects mitochondrial metabolism under conditions such as ischemia. Mildronate also acted as a neuroprotective agent in an azidothymidine-induced mouse model of neurotoxicity, as well as in a rat model of Parkinson's disease. These observations suggest that mildronate may stimulate processes involved in cell survival and change expression of proteins involved in neurogenic processes. The present study investigated the influence of mildronate on learning and memory in the passive avoidance response (PAR) test and the active conditioned avoidance response (CAR) test in rats. The CAR test employed also bromodeoxyuridine (BrdU)-treated animals. Hippocampal cell BrdU incorporation was then immunohistochemically assessed in BrdU-treated, CAR-trained rats to identify proliferating cells. In addition, the expression of hippocampal proteins which could serve as memory enhancement biomarkers was evaluated and compared to non-trained animals' data. These biomarkers included glutamic acid decarboxylase 65/67 (GAD65/67), acetylcholine esterase (AChE), growth-associated protein-43 (GAP-43) and the transcription factor c-jun/activator protein-1 (AP-1). The results showed that mildronate enhanced learning/memory formation that coincided with the proliferation of neural progenitor cells, changing/regulating of the expression of biomarker proteins which are involved in the activation of glutamatergic and cholinergic pathways, transcription factors and adhesion molecule.

The data from our study suggest that mildronate may be useful as a possible cognitive enhancer for the treatment of patients with neurodegenerative diseases with dementia.



Mildronate Dosage
Interestingly, the neuroprotective dose of Mildronate is much lower than the usual dose.




Summary. This review for the first time summarizes the data obtained in the neuropharmacological studies of mildronate, a drug previously known as a cardioprotective agent. In different animal models of neurotoxicity and neurodegenerative diseases, we demonstrated its neuroprotecting activity. By the use of immunohistochemical methods and Western blot analysis, as well as some selected behavioral tests, the new mechanisms of mildronate have been demonstrated: a regulatory effect on mitochondrial processes and on the expression of nerve cell proteins, which are involved in cell survival, functioning, and inflammation processes. Particular attention is paid to the capability of mildronate to stimulate learning and memory and to the expression of neuronal proteins involved in synaptic plasticity and adult neurogenesis. These properties can be useful in neurological practice to protect and treat neurological disorders, particularly those associated with neurodegeneration and a decline in cognitive functions.

Concluding Remarks

The obtained data give a new insight into the influence of mildronate on the central nervous system.

This drug shows beneficial effects in the regulation of cell processes necessary for cell integrity and survival, particularly by targeting mitochondria and by stabilizing the expression of proteins involved in neuroinflammation and neuroregeneration. These properties can be useful in neurological practice to protect and treat neurological disorders, such as Parkinson’s disease, diabetic neuropathies, and ischemic stroke. Moreover, because mildronate improves learning and memory, one may suggest mildronate as a multitargeted neuroprotective/ neurorestorative drug with its therapeutic utility as a memory enhancer in cognitive impairment conditions, such as neurodegenerative diseases, schizophrenia, and other pathologies associated with a decline in awareness.

The present review summarizes our previously obtained data which demonstrated the influence of mildronate on mitochondrial processes and the expression of nerve cell proteins involved in the essential pathways for cell survival and functioning. Besides, the effectiveness of mildronate at much lower doses of 20 and 50 mg/kg in comparison with the traditionally recommended doses typical for cardioprotection (100 and 200 mg/kg) has been demonstrated.



Bypass the need for Complex 1 by ketosis?

Almost all the research on mitochondrial disease assumes that you want to convert glucose to ATP.
If a person has an inability to produce enough complex 1 they might be better off switching from glycolysis (glucose as fuel) to ketosis (ketones from fat as fuel).
There are posts in this blog describing the ketogenic diet, which has been widely used for decades to treat epilepsy.

Ketosis is a metabolic state in which some of the body's energy supply comes from ketone bodies in the blood, in contrast to a state of glycolysis in which blood glucose provides most of the energy.

Ketosis is a nutritional process characterized by serum concentrations of ketone bodies over 0.5 mM, with low and stable levels of insulin and blood glucose. It is almost always generalized with hyperketonemia, that is, an elevated level of ketone bodies in the blood throughout the body. Ketone bodies are formed by ketogenesis when liver glycogen stores are depleted (or from metabolising medium-chain triglycerides). The main ketone bodies used for energy are acetoacetate and β-hydroxybutyrate, and the levels of ketone bodies are regulated mainly by insulin and glucagon. Most cells in the body can use both glucose and ketone bodies for fuel, and during ketosis, free fatty acids and glucose synthesis (gluconeogenesis) fuel the remainder.

As is often the case, opinion is mixed on the ketogenic diet and mitochondrial disorders. It seems to make some people better and have no effect on others.  This is likely because they do not have precisely the same mitochondrial disorder.



2.6. Dietary manipulations

Several approaches based on dietary measures have been attempted, with controversial results. Ketogenic diet (KD), i.e. a high-fat, low-carbohydrate diet, has been proposed to stimulate mitochondrial beta-oxidation, and provide ketones, which constitute an alternative energy source for the brain, heart and skeletal muscle. Ketone bodies are metabolized to acetyl-CoA, which enters the Krebs cycle and is oxidized to feed the RC and ultimately generate ATP via OXPHOS. This pathway partially bypasses complex I via increased synthesis of succinate, which donates electrons to the respiratory chain via complex II. Increased ketone bodies have also been associated with increased expression of OXPHOS genes, possibly via a starvation-like response [80]. Starvation is a stressing condition to the cell, which results in activation of many transcription factors and cofactors (including SIRT1, AMPK, and PGC-1α) that ultimately increase mitochondrial biogenesis [80]. KD reduced the mutation load of a heteroplasmic mtDNA deletion in a cybrid cell line from a Kearns–Sayre syndrome patient [81], was shown to increase the expression levels of uncoupling proteins and mitochondrial biogenesis in the hippocampus of mice and rats [82] and [83], and increased mitochondrial GSH levels [84] in rat brain. These phenomena could contribute to explain the anticonvulsant effects of KD. In a preclinical trial on the deletor mouse, KD slowed the progression of mitochondrial myopathy [85]. However, other reports showed that KD can have the opposite effect, and worsens the mitochondrial defect invivo, for instance in the Mterf2−/− [86], or the Mpv17–/−mouse models [87].

Similar to KD, a high fat diet (HFD) was shown to have a protective effect on fibroblasts with complex I deficiency and be effective in delaying the neurological symptoms of the Harlequin mouse, a model of partial complex I defect associated with a homozygous mutation of AIFM1, encoding the mitochondrial apoptosis inducing factor [88].

Similar results could in principle be achieved using other compounds that release succinate in mitochondria. An example is triheptaoin, an anaplerotic compound inducing a rapid increase of plasmatic C4- and C5-ketone bodies, the latter being a precursor of propionyl-CoA, which is then converted into succinyl-CoA. Treatment with triheptaoin has been reported to dramatically improve cardiomyopathy in patients with VLCAD deficiency and myopathic symptoms in CPT2 deficiency patients [89] and [90]. 



Ketogenic diet

The ketogenic diet is a high-fat diet that effectively treats some forms of medically refractory epilepsy [7,8, Class I]. Recent animal research has suggested that the ketogenic diet may be beneficial in optimizing mitochondrial function [9, Class III].
Because many mitochondrial disease patients have secondary fatty acid oxidation disorders, there are limited data on use and safety of the ketogenic diet in patients with these conditions. Only a single report has looked at the lack of efficacy of the ketogenic diet in children with electron transport chain defects and intractable seizures [10, Class IV].
The ketogenic diet is the standard of care for pyruvate dehydrogenase deficiency, but it is contraindicated in patients with known fatty acid oxidation disorders and pyruvate carboxylase deficiency.

Experimental Therapies

Highlights


o   At present there is no effective cure for mitochondrial diseases.

o   Generalist and tailored therapies are emerging at the pre-clinical level.

o   Some therapies are effective in disease models and ready for translation to patients.

o   Other approaches warrant more work at the pre-clinical level.


Conclusion
Some people’s autism does indeed appear to have been solely caused by the lack of mitochondrial enzymes.  These dysfunctions can be inherited or acquired.

As Dr Kelley suggests, a baby might be born with a 50% reduction in complex 1 and develop normally. Following a viral infection, or other insult, before the brain has substantially matured a further reduction in complex 1 occurs and this tips the balance to where mitochondria cannot function sufficiently. Siblings may have exactly the same biochemical markers, but continue normal development because they avoided the damaging insult that triggered regression at a critical point in the brain’s maturation.
The data does point to mitochondrial dysfunction being present beyond just those with regressive autism, so a little extra complex 1 may be in order for them too.

Of the five enzyme complexes, complex 1 appears to be the most important because it is “rate limiting”, meaning it is usually the enzyme with the least unused capacity.  It becomes the bottleneck in the energy production chain. Many other diseases and aging feature a decline in complex 1 which may account for some people’s loss of cognitive function.
Is mildronate a carnitine analog with better bioavailability? Are its cognitive enhancing effects due to increased blood flow, improved complex 1 availability or perhaps both?  We can only wait till the Latvians do some experiments on schizophrenia and autism.  The good news is that the dose at which the mitochondrial effects occur is five times less than the anti-ischemia dose.

I can see that the dose for athletes is twice the dose for ischemia. So it would seem that tennis players who have used mildronate for ten years, at ten times the mitochondrial dose, might provide some useful safety information.



As you can see from the packaging, the drug must be popular with cyclists too.


Suggested further reading, or indeed re-reading:



Richard I. Kelley, MD, PhD
Division of Metabolism, Kennedy Krieger Institute Department of Pediatrics, Johns Hopkins Medical Institutions