Showing posts with label Epicatechin. Show all posts
Showing posts with label Epicatechin. Show all posts

Wednesday, 1 November 2017

OAT3 inhibitors for Bumetanide - Probenecid, but also Aspirin, Chlorogenic acid (Coffee), Epicatechin (Cocoa, Cinnamon) and more.

Today’s post is about OAT3, highlighted by the green lines.
The interventions reduce renal excretion and raise plasma
concentration rather than directly improving transport across the BBB

Today’s post is a collaboration. Our reader Ling pointed out research trying to boost the bioavailability of bumetanide using something clever called an OAT3 inhibitor.  This would reduce the rate at which the body excretes bumetanide and thus potentially improve its therapeutic effect.
Petra, our reader from Greece, pointed out that in her son Bumetanide seemed to work better when taken with Greek coffee and that that Greek Grandpas like to take their diuretics with a steaming Greek coffee.
Most people, me included, automatically think caffeine when someone mentions coffee.
So I assumed that caffeine might be an OAT3 inhibitor and I did make some experiments on that basis. There is no research data to support caffeine as an OAT3 inhibitor.
Recently I was again looking for other potential Bumetanide boosters.  The obvious one is called Probenecid.  Probenecid is used to treat gout because it lowers uric acid.
Aspirin has some odd effects; low dose aspirin will raise uric acid, but high dose aspirin will lower it. Aspirin is an OAT3 inhibitor.
OATs are a very niche subject, to add to the confusion sometimes you are better looking for SLC22A8, the gene that encodes the transporter. 
There was an earlier post on this subject, which showed that many NSAIDs inhibit OAT3, including Knut’s favourite Ponstan. They are not so well suited to continued use.

At the end of my little investigation I figured it out; there are many OAT3 inhibitors available, including some in your kitchen.  

Key points on OAT3 (Organic Anion Transporter 3)
If you want to increase the peak concentration and indeed the half-life of a drug that is excreted from the body by OAT3 (organic anion transporter 3), an OAT inhibitor is what you need.
The drug Probenecid is by far the best known OAT3 inhibitor and it is very potent. It has long been to boost the performance of penicillin type antibiotics to treat tough bacterial infections.
Probenecid, if available, may very well be the ideal bumetanide booster.
For adults a simple option is Greek/Turkish coffee. I see little downside as long as you can handle the caffeine. The Greeks live a long time and drink plenty of coffee.
For those who do not like caffeine you can go to active components within the coffee, which seem to be the chlorogenic acids (1,3- and 1,5-dicaffeoylquinic acid). They are sold as a weight loss supplement, the long established version is the French-made Svetol, but there are now others. They still contain 2- 3% caffeine.
Epicatechin, found in cinnamon, dark chocolate and high flavanol cocoa is another OAT3 inhibitor. Cocoavia, made by Mars, is used by some readers of this blog. Cocoa flavanols do clever things with nitric oxide (NO) and have been shown to improve mild cognitive impairment (MCI) and heart health by improving blood vessel elasticity.
Catechins are flavanols belonging to a family of closely related compounds, such as epicatechin, epigallocatechin, epicatechin gallate (EGC), and epigallocatechin gallate (EGCG). They are all slightly different. Catechin itself is not an OAT3 inhibitor; EGCG may or may not be.
Low dose aspirin is likely the cheapest OAT3 inhibitor. It also increases peripheral circulation, which could benefit some. Low dose aspirin has the downside of a small bleeding risk, mainly in old people, and there is a risk of Reye’s syndrome if given during/after a viral infection.
I think for adults a Greek coffee may be the best. For people who have a profound benefit from Bumetanide, I think they should look into Probenecid.
Personally I think Svetol is worth a try.
Coffee that has been extensively processed (just as we saw with cocoa) may not have the same chlorogenic acid content as the more gritty coffee used in the Balkans. Coffee consumption is actually associated with many neurological benefits, reducing the incidence of Parkinson’s and Alzheimer’s; the common mistake in research is the assumption that the effect must be from caffeine.

The health effects of decaffeinated Coffee
My eureka moment in this post was reading about gout and coffee and then decaffeinated coffee. 

So then it was a question of finding what in coffee could be the OAT3 inhibitor. At which point I found a very insightful paper that tells you everything, once you realise that:

Coffee = chlorogenic acids  = 1,3- and 1,5-dicaffeoylquinic acid

Five compounds, 1,3- and 1,5-dicaffeoylquinic acid, ginkgolic acids (15 : 1) and (17 : 1), and epicatechin, significantly inhibited hOAT3 transport under similar conditions

3.2. Inhibition of hOAT3 by Natural Anionic Compounds and Flavonoids

Human OAT3 expressing cells showed about 4-fold greater accumulation of ES as compared to background control cells ( versus  pmol mg 10  , resp.). Similar to hOAT1, hOAT3-mediated ES uptake was completely (>96% inhibition) blocked by probenecid (Figure 4). Five of the compounds, 1,3- and 1,5-dicaffeoylquinic acid, epicatechin, and ginkgolic acids (15 : 1) and (17 : 1), significantly inhibited hOAT3-mediated transport at 50-fold excess (Figure 4). 1,3-Dicaffeoylquinic acid and ginkgolic acid (17 : 1) exhibited 41% inhibition, while 30–35% reduction of hOAT3-mediated ES uptake was observed for 1,5-dicaffeoylquinic acid, epicatechin, and ginkgolic acid (15 : 1). Catechin, 18β-glycyrrhetinic acid, and ursolic acid failed to produce significant inhibition. Based on the level of inhibition observed, values for all of these compounds would be greater than 50 μM, much higher than clinically relevant concentrations (Table 1). Therefore, further dose-response studies were not performed.

Lay off the Lycopene?
Lycopene does the opposite of what we want. Too much lycopene may lower the effectiveness of a drug that is excreted via OAT3. 

2.29. Lycopene

Lycopene is a carotenoid pigment found in tomato [94]. Lycopene from dietary sources has been shown to reduce the risk of some chronic diseases including cancer and cardiovascular disorders [95]. The administration of lycopene significantly normalized the kidney function and antioxidant status of CSP-treated animals. Furthermore, lycopene also increased the expression of the organic anion and cation transporters (OAT and OCT, resp.) including OAT1, OAT3, OCT1, and OCT2 in the renal tissues [9698]. In addition, lycopene also decreased the renal efflux transporters (multidrug resistance-associated protein [MRP]-2 and MRP4) levels and induced Nrf2 activation, which activated the antioxidant defense system [99]. Furthermore, lycopene protected against CSP-induced renal injury by modulating proapoptotic Bax and antiapoptotic Bcl-2 expressions and enhancing heat shock protein (HSP) expression [97].                                                                                                                  

I actually started out this post by looking at what dose of aspirin might be effective in inhibiting OAT3.  We do know that Aspirin is indeed an OAT3 inhibitor.  

I did find the answer, but along the way you do end up having to look at uric acid. 
Uric acid is taken up by OAT1 and OAT3 from the blood and reabsorbed into renal tubular cells via URAT1 Uric acid is taken up by OAT1 and OAT3 from the blood and reabsorbed into renal tubular cells via URAT1Uric acid is taken up by OAT1 and OAT3 from the blood and reabsorbed into renal tubular cells via URAT1. 
Uricosuric drugs increase the excretion of uric acid in the urine, thus reducing the concentration of uric acid in blood plasma. 
In general, uricosuric drugs act on as urate transporter 1 (URAT1). URAT1 is the central mediator in the transport of uric acid from the kidney into the blood.  By their mechanism of action, some uricosurics (such as  probenecid) increase the blood plasma concentration of certain other drugs and their metabolic products  – this is their effect on OAT3.
Probenecid is a medication that increases uric acid excretion in the urine.
Atorvastatin is a so-called secondary uricosuric. High dose aspirin should also be called a secondary uricosuric.
Antiuricosuric drugs raise serum uric acid levels and lower urine uric acid levels. These drugs include all diuretics and low dose aspirin. 
Low dose aspirin inhibits OAT1 and OAT3 which reduces urate secretion, but high dose aspirin inhibits URAT1 and reduces urate re absorption. This is sometimes known as the biphasic effect.
So low dose aspirin will increase plasma uric acid, but high dose aspirin has the same effect as Probenecid, it lowers plasma uric acid levels.
So Aspirin and Probenecid both affect URAT1 and OAT3. 

At what dose is Aspirin an OAT3 inhibitor?
If we just want aspirin to inhibit OAT3 and not inhibit URAT1, what dose is effective? Fortunately this has been answered in the research. The typical low dose of aspirin (75mg) used preventatively in older people is OAT3 inhibiting, it raises plasma uric acid.  


Salicylic acid and its derivatives are the most prescribed analgesic, antipyretic, and anti-inflammatory agents. Salicylates have a “paradoxical effect” on the handling of uric acid by the kidney. The action of salicylates on uric acid excretion depends on the dose of salicylates. At doses of less than 2.5 g/day, salicylates cause the retention of uric acid by blocking the tubular secretion of uric acid, while at dose of higher than 3 g/day, they cause increased urinary excretion of uric acid [70]. Mini-dose aspirin, even at a dosage of 75 mg/day, caused a decrease in uric acid excretion and raised serum uric acid level [71]. It has been suggested that the “paradoxical effect” of salicylate can be explained by two modes of salicylate interaction with URAT1: (1) acting as an exchange substrate to facilitate uric acid reabsorption, and (2) acting as an inhibitor for uric acid reabsorption [72]. Low dose of salicylate interact with OAT1/OAT3, the uric acid secreters [73].

Low dose aspirin leads to decreased renal excretion of uric acid and raised serum uric acid levels, which can cause a gout attack in those predisposed to this condition.
High doses of aspirin lower serum uric acid concentration.

Reye’s Syndrome
In children aspirin is very rarely used because of the risk of Reye’s syndrome. Reye’s syndrome causes severe liver and brain damage. It is a type of severe mitochondrial failure that can occur after a viral infection like flu or chickenpox, but it almost only occurs when aspirin has been prescribed. Nobody knows for sure the exact mechanism of the disease.
So do not give aspirin to children with a viral infection.  We already know to avoid paracetamol/acetaminophen (Tylenol in the US) in babies/children and people with autism. Paracetamol/acetaminophen depletes the body’s key antioxidant GSH. 
If someone overdoses on Paracetamol/acetaminophen you give them a high dose of NAC to prevent death. 

Given how long it takes to develop new drugs, I think that improving the pharmokinetics of bumetanide is a pretty obvious thing to do. 
Diamox is an OAT3 inhibitor and our reader Agnieszka found it beneficial only when administered along with Bumetanide.
Strong coffee is an OAT3 inhibitor and this was found to enhance bumetanide by Petra’s son with Asperger’s.
Cinnamon which contains epicatechin, another OAT3 inhibitor, did seem to be helpful in Monty who also takes bumetanide.
I suspect Diamox may be the most potent OAT3 inhibitor of those three
The interesting OAT3 inhibitors seem to be:-

·        Probenecid

·        Low dose aspirin

·        Epicatechin (cocoa, cinnamon ..)

·        Chlorogenic acids (coffee and decaffeinated green coffee extracts) 

Cinnamon, high flavanol cocoa and indeed coffee (minus the caffeine) have numerous health benefits.
Note that Catechin has no effect on OAT3. EGCG was not tested but in other studies has been shown it does affect.

The logical next step would be to improve bumetanide transport across the blood brain barrier.

Monday, 2 November 2015

Brain Hypoperfusion in Autism & Cocoa

Today’s post is simpler than many earlier ones and is actionable.

A known feature of many neurological conditions like Alzheimer’s and dementia is reduced blood flow to certain parts of the brain.  In the medical jargon this is called hypoperfusion.

This reduced blood flow is also present in autism and is measurable by MRI.

We encountered epicatechin in early posts on cocoa flavanols.  It would seem that one of epicatechin’s many effects is to increase cerebral blood flow. 

Two chocolate companies, Mars (Cocoavia) in the US and Barry Callebaut (ACTICOA) in France, have developed high flavanol cocoa.  10 g of their cocoa contains about 1 g of flavanols and produces cognitive benefits; even a quarter of this dose gives the cardiovascular benefits.  Mars, in particular, are funding a great deal of research and have committed to a five year project with Harvard.  The high flavanol products are available today.

Brain Perfusion Anomalies in Autism

While most research focuses on Alzheimer’s and other types of cognitive impairment and memory loss, there are studies on brain perfusion in autism.

Autism is a severe developmental disorder, the biological mechanisms of which remain unknown. Hence we conducted this study to assess the cerebral perfusion in 10 children with autism and mental retardation. Five age matched normal children served as controls. These cases were evaluated by single photon emission computed tomography (SPECT) using Tc-99m HMPAO, followed by segmental quantitative evaluation. Generalized hypoperfusion of brain was observed in all 10 cases as compared to controls. Frontal and prefrontal regions revealed maximum hypoperfusion. Subcortical areas also indicated hypoperfusion. We conclude that children with autism have varying levels of perfusion abnormities in brain causing neurophysiologic dysfunction that presents with cognitive and neuropsychological defects.
Significant hypoperfusion was observed at cortical and subcortical areas of brain in autistic subjects, suggesting that the structural abnormalities
of these brain areas may result in reduced cortical activity, thus causing dysfunction of these brain areas, and eventually producing some of the
emotional and behavioral disorders usually described in autistic subjects. These SPECT findings may help to explain several behavioral features of autism, such as impulsive and aggressive behaviours (to self and others), motor disinhibition (such as stereotypic and manneristic movements and echophenomena), and deficits in planning, sequencing and attention.

Abnormal regional cerebral blood flow in childhood autism

Neuroimaging studies of autism have shown abnormalities in the limbic system and cerebellar circuits and additional sites. These findings are not, however, specific or consistent enough to build up a coherent theory of the origin and nature of the brain abnormality in autistic patients. Twenty-three children with infantile autism and 26 non-autistic controls matched for IQ and age were examined using brain-perfusion single photon emission computed tomography with technetium-99m ethyl cysteinate dimer. In autistic subjects, we assessed the relationship between regional cerebral blood flow (rCBF) and symptom profiles. Images were anatomically normalized, and voxel-by-voxel analyses were performed. Decreases in rCBF in autistic patients compared with the control group were identified in the bilateral insula, superior temporal gyri and left prefrontal cortices. Analysis of the correlations between syndrome scores and rCBF revealed that each syndrome was associated with a specific pattern of perfusion in the limbic system and the medial prefrontal cortex. The results confirmed the associations of (i) impairments in communication and social interaction that are thought to be related to deficits in the theory of mind (ToM) with altered perfusion in the medial prefrontal cortex and anterior cingulate gyrus, and (ii) the obsessive desire for sameness with altered perfusion in the right medial temporal lobe. The perfusion abnormalities seem to be related to the cognitive dysfunction observed in autism, such as deficits in ToM, abnormal responses to sensory stimuli, and the obsessive desire for sameness. The perfusion patterns suggest possible locations of abnormalities of brain function underlying abnormal behaviour patterns in autistic individuals.

Cerebral Hypoperfusion and HBOT?

One therapy proposed to treat Cerebral Hypoperfusion in autism is hyperbaric oxygen therapy (HBOT).  Some proponents go as far as to link specific areas of the brain to specific autistic features as below.

The mainstream view, among those using HBOT for other conditions, is that it would not help stimulate increased blood flow in autistic brains.  But there are proponents of the therapy like Rossignol.

You may have realized that the science exists to test, once and for all, whether HBOT can improve cerebral blood flow in autism.  It just takes two visits to an MRI.

I did see a report about a US neurologist who showed via MRI that the cerebral blood flow of his autistic patient improved using HBOT and he tried to use this to get access to the further HBOT on insurance.

Hypoperfusion in Alzheimer’s, Dementia  and Cognitive Impairment

Reduced cerebral blood flow is a marker of incipient dementia.  I expect one day this might even be used to trigger preventative therapy.

Cerebral hypoperfusion and clinical onset of dementia: the Rotterdam Study.


Cerebral blood flow (CBF) velocity is decreased in patients with Alzheimer's disease. It is being debated whether this reflects diminished demand because of advanced neurodegeneration or that cerebral hypoperfusion contributes to dementia. We examined the relation of CBF velocity as measured with transcranial Doppler with dementia and markers of incipient dementia (ie, cognitive decline and hippocampal and amygdalar atrophy on magnetic resonance imaging) in 1,730 participants of the Rotterdam Study aged 55 years and older. Cognitive decline in the 6.5 years preceding CBF velocity measurement was assessed with repeated Mini-Mental State Examinations in nondemented subjects (n = 1,716). Hippocampal and amygdalar volumes were assessed in a subset of 170 nondemented subjects. Subjects with greater CBF velocity were less likely to have dementia. Furthermore, in nondemented subjects, greater CBF velocity was related to significantly less cognitive decline over the preceding period (odds ratio per standard deviation increase in mean CBF 0.74 [95% confidence interval, 0.58-0.98]) and larger hippocampal and amygdalar volumes. A low CBF is associated with dementia, but also with markers of incipient dementia. Although we cannot exclude that this is caused by preclinical neurodegeneration leading to hypoperfusion, it does suggest that cerebral hypoperfusion precedes and possibly contributes to onset of clinical dementia.

Vascular dementia

Vascular dementia is the second-most-common form of dementia after Alzheimer's disease.  It is a much simpler condition, it is dementia caused by problems in the supply of blood to the brain, typically by a series of minor strokes.

The incidence peaks between the fourth and the seventh decades of life and 80% will have a history of hypertension. Patients develop progressive cognitive, motor and behavioural signs and symptoms.

Blood pressure rises with aging and the risk of becoming hypertensive in later life is considerable

It would seem that you could treat hypertension and vascular dementia with the same preventative therapy.  See the clinical trial on treating vascular aging with Cocoa, later in this post.

It has also been suggested that endothelial dysfunction and vascular inflammation may also contribute to increased peripheral resistance and vascular damage in hypertension. 

In essence you want to control peripheral resistance and before it is too late.  It really is a case of “a stitch in time saves nine”.

The research done in to peripheral resistance / vascular stiffness can be re-purposed to help us treat brain hypoperfusion.  In autism we may have Brain Hypoperfusion, but without high blood pressure (hypertension).

Increased vascular stiffness, endothelial dysfunction, and isolated systolic hypertension are hallmarks of vascular aging. Regular cocoa flavanol (CF) intake can improve vascular function in healthy young and elderly at-risk individuals. However, the mechanisms underlying CF bioactivity remain largely unknown. We investigated the effects of CF intake on cardiovascular function in healthy young and elderly individuals without history, signs, or symptoms of cardiovascular disease by applying particular focus on functional endpoints relevant to cardiovascular aging. In a randomized, controlled, double-masked, parallel-group dietary intervention trial, 22 young (<35 years) and 20 elderly (5080 year) healthy, male non-smokers consumed either a CF-containing drink (450 mg CF) or nutrientmatched, CF-free control drink bi-daily for 14 days.
The primary endpoint was endothelial function as measured by flow-mediated vasodilation (FMD). Secondary endpoints included cardiac output, vascular
stiffness, conductance of conduit and resistance arteries, and perfusion in the microcirculation. Following 2 weeks of CF intake, FMD improved in young (6.1±0.7 vs. 7.6±0.7 %, p<0.001) and elderly (4.9 ± 0.6 vs. 6.3 ± 0.9 %, p < 0.001).
Secondary outcomes demonstrated in both groups that CF intake decreased pulse wave velocity and lowered total peripheral resistance, and increased arteriolar and microvascular vasodilator capacity, red cell deformability, and diastolic blood pressure, while cardiac output remained affected. In the elderly, baseline systolic blood pressure was elevated, driven by an arterial-stiffness-related augmentation.
CF intake decreased aortic augmentation index (9 %) and thus systolic blood pressure (7 mmHg;

Cocoa Flavanols

I did write an earlier post about the various benefits of Cocoa Flavanols. 

Here is a very good review paper:-

Norman Hollenberg, at Harvard, has been an advocate of high flavanol cocoa for decades.  Here is one of his papers.

Using functional MRI, the following study measures the effect on brain blood flow, before and after taking a high flavanol cooca drink

There is now good evidence that the acute benefits for cognitive function and blood flow exerted by cocoa flavanol consumption peak approximately 90120 min postconsumption (Schroeter et al. 2006; Francis et al. 2006; Scholey et al. 2010; Field et al. 2011); however, it is presently unclear whether separate chronic mechanisms exists following cumulative consumption over several weeks and months, or indeed whether chronic consumption enhances the effectiveness of acute mechanisms in a cumulative fashion. Despite several plausible mechanisms for increased neuronal activity (as described above), it remains to be seen whether a single cocoa flavanol dose-induced increase in CBF is associated with concomitant benefits in cognitive performance in the immediate postprandial period. More broadly, recent reviews of acute interventions and epidemiological surveys provide good evidence that flavonoids and their subclasses are beneficial for cognitive function

In conclusion, the present findings support the hypothesis that flavanol-rich cocoa beverages are associated with increased CBF within a 2-h post-prandial time frame. More specifically, increased brain perfusion following the HF drink relative to the LF drink was observed in the anterior cingulate cortex and a region in the left parietal lobe. These data add to the substantial body of literature demonstrating that flavanol consumption is beneficial for peripheral and cerebral vascular function and thus for maintaining, protecting and enhancing cardiovascular health.

Does High Flavanol Cocoa have an effect in Autism?

This is probably the question you have been asking yourself.

I did acquire some ACTICOA, high flavanol cocoa some time ago.  I was wondering how I was going to administer enough of it to make a trial.  In the trials on improving memory in older adults 10g a day was needed.

While adding it to milk seems an obvious choice, Hollenberg suggests that the milk may neutralize the flavanols.  This is true with black tea; once you add milk you lose its healthy antioxidant properties.

In the end I choose to add 5ml to the breakfast broccoli powder and water concoction and mix with a frappe mixer.  Monty, aged 12 with ASD, was the ever willing test subject.

Two and a half hours later there was unprompted laughter and smiling.  This is repeated each time I give the ACTICOA  cocoa.

According to the literature, the peak level of epicatechin occurs 2 to 3 hours after consuming cocoa.

Then I tried a regular raw cocoa powder at the same dose; no laughter.

So I conclude that ACTICOA is indeed different to regular non-alkalized cocoa powder.  The more common alkalized cocoa has virtually no flavanols at all, and this is what is used to make most chocolate and is sold in supermarkets as "cocoa".

There are potentially other sources of epicatechin, but you really want a reliable standardized product.  If you live in the US/Canada this is easy; you can buy the Cocoavia product from Mars.  It is not cheap if you want 1g of flavanols a day.

The literature does suggest that there is a cumulative effect of taking epicatechin and Hollenberg has documented that regular consumption of unprocessed cocoa (rich in flavanols) is associated with numerous health benefits, particularly related to blood flow (strokes, heart attacks, endothelial dysfunction, cholesterol etc.)

Since Mars are now funding considerable research into the health benefits of these flavanols, I did think of suggesting they look at autism.

They could take a group of people with autism, measure their IQ and then score their autism using one of the standard scales.  Then off to the MRI to measure blood flow and velocity in different parts of the brain.

Give half of the test subjects a daily high flavanol drink and the other half a low flavanol drink.  After three months, repeat the IQ test, autism test and measure blood flow again via MRI.

I suspect that reduced blood flow/hypoperfusion would be more present in those with lower IQ and that they might show improved IQ at the end of the trial.  I suspect that in terms of autism, most would show an improvement on the high flavanol treatment.

I would like to think that after three months, blood flow/velocity would have increased.

You could then repeat on people with Down Syndrome and more general MR/ID.

Thursday, 3 September 2015

Gene Silencers and Enhancers in Autism; plus Epicatechin, MOCOS, Ferritin and Oxidative Stress (GR, GPx, GCL, GCLM)

The original idea of this blog was to try to keep complicated things as simple as possible, so at times things may get over-simplified.  

This post starts out simple and then gets rather involved in oxidative stress.

When people think about genes, they are nearly always thinking about the “blueprints” that are encoded on your DNA.  As it turns out only about 5% of your DNA is dedicated to this function; this 5% is contained in the exome.

Much autism research is dedicated to finding faulty “blueprints” that might account for autism.  There are now several hundred so called “autism genes”, where an error in the “blueprints”, means that the associated protein is not produced to its intended specification.

We also have seen that genetic defects just lead to a possibility of something going wrong.  A “faulty gene” creates the possibility of a specific dysfunction happening, it does not mean 100% that it will happen. 

Partial dysfunctions and partial deficiency

We also saw that even when a single gene dysfunction, like for fragile-X, occurs it does not always cause a catastrophic failure, rather it produces a spectrum from mild to severe.

This point is important since it seems in autism there can often be “partial dysfunctions” leading to “partial deficiencies”.  This is just a less severe form of the “rare” total dysfunctions.  The growing list of examples includes partial biotinidase deficiency, partial glutathione reductase deficiency and partial glutathione peroxidase deficiency.  Today we will also encounter ferritin (iron storage) partial deficiencies.  In a future post we will look the vitamin B12 partial dysfunction that occurs in about a quarter of schizophrenia and autism cases.

This then leads us to the subject of gene expression, which means how much, where, when and how often a gene is turned “on”.  This is actually what really matters, since even perfectly good genes, when over-expressed, can do great damage.  We saw that in the case of Down Syndrome there is about 50% over expression in up to 300 genes.  In the case of Down Syndrome the reason for this overexpression lies in the exome.  In effect there is a double set of blueprints for those 300 genes.

Within the remaining 95% of your DNA are so-called enhancers and silencers.  Their job is to determine which genes are turned on (enhancers) or turned off (silencers) in which part of the body.  So a gene might encode a calcium channel, but that calcium channel should only be in certain parts of the body and only to a certain degree.  We need the correct clean blueprint and we need it applied in the right part of the body and only to the desired extent.

I was very pleased to see that some scientists have started to look at the role of enhancers in autism.  I have already noticed that some substances that are known to affect gene expression are particularly effective in autism.  This suggests to me that in some types of autism, the problem may actually be simply in gene expression rather than any faulty genetic “blueprint”.

Now the science of enhancers and particularly silencers is still at the emerging stage, but the research showed that in at least 100 locations, there were significant anomalies in those with autism.

This is an easy to read summary of the research paper below.


Despite major progress in identifying enhancer regions on a genome-wide scale, the majority of available data are limited to model organisms and human transformed cell lines. We have identified a robust set of enhancer RNAs (eRNAs) expressed in the human brain and constructed networks assessing eRNA-gene coexpression interactions across human fetal brain and multiple adult brain regions. Our data identify brain region-specific eRNAs and show that enhancer regions expressing eRNAs are enriched for genetic variants associated with autism spectrum disorders.

We also have the removable markers on the 5% of DNA that cause epigenetic changes.  This is another way of turning on or off specific genes.  These markers can be caused by environment factors like smoking, or even stress, these markers are potentially both removable and inheritable.     

The emerging science of Proteomics is the study of gene expression itself, so it is measuring all the proteins that the genes actually produced.

Limits of Genetic Testing

So while in some cases genetic testing of the 5% of DNA usually examined may indeed be useful, if your problem was in the other 95% of DNA it will not help.

To be useful in autism you would need to measure gene expression in the brain or the local activity of the enhancers/silencers, since it varies throughout the body.  In the Australian study above they measured the enhancer activity in the brain, by looking for the special enhancer molecules the enhancers produce.

This is all way beyond the scope of this blog.

However when I see “safe” substances like Sulforaphane, Epicatechin and even statins that are known to affect the expression of multiple genes, I take note. 

Steroids also affect gene expression, but great care has to be taken with steroids.

Statins have numerous interesting effects in the brain and in cancer cells.  In autism they have an effect on PTEN and BCL2 for example.

The observed impact of pravastatin on gene expression may explain the pleiotropic effects of statins when they are used as adjuvants in chemotherapy and suggests impact on gene expression as a possible cause of side effects from statin use.

As pointed out in the last paper, changing gene expression can be bad as well as good.  It all depends where you are starting from and what genes you want to enhance/silence.

Other therapies to modify gene expression

Today’s scientific knowledge does not always allow us to target the expression of specific genes, this very much remains future science.

However, the remarkable effects of some substances, in some people, does suggest some options.  As is often the case this takes us back to oxidative stress, which does seem to affect many conditions and is quite well studied. There is no shortage of anecdotal evidence.

We know from the research that oxidative stress is ever-present in autism and that people with autism are particularly sensitive to it.

One substance previously mentioned in this blog, epicatechin, is known to change the expression of many genes including STAT1, MAPKK1, MRP1, and FTH1, which are involved in the cellular response to oxidative stress.


Rather off subject the FTH1 gene encodes the heavy subunit of ferritin, the major intracellular iron storage protein.

Children with autism spectrum disorders had significantly lower ferritin levels compared with controls
Within the autism spectrum disorders population, median ferritin levels were significantly lower in patients with poor sleep efficiency (7 ng/mL) versus those with normal sleep efficiency (29 ng/mL) (P = 0.01).

Low ferritin would indicate an iron storage problem and likely anemia/anaemia

Low ferritin has many effects, including surprisingly, poor sleeping patterns.
Is it such a surprise that a cup of cocoa (epicatechin) before bed used to be given to ensure a good night’s sleep?  (all via FTH1, I presume)

Perhaps poor sleep in autism is just another consequence of oxidative stress?


In the recent paper on MOCOS:-

I noted that:-

Furthermore, we found that MOCOS misexpression induces increased oxidative-stress sensitivity.

MOlybdenum COfactor Sulfurase (MOCOS), is an enzyme involved in purine metabolism and a newly identified player in ASD. MOCOS appears to be downregulated in autism and this has multiple effects, one being increased sensitivity to oxidative stress.

Seemingly unknown to the French MOCOS researchers, there already is a therapy:-

Since I do not have any of the above biosynthetic precursor at hand, but I do have high flavanol cocoa in the kitchen, it is time to look again at epicatechin.


There are two very similar substances catechin and epicatechin; both are flavonoids.  Both affect gene expression and both seem to have numerous good properties.

Epicatechin is found in large quantities in mildly processed cocoa, which catechin in found in large quantities in certain types of Chinese tea.

We saw in an earlier post that Mars, the chocolate company, has invested substantially in the science of cocoa and its flavonoids.  They have just signed a 5 year research contract with Harvard.

Catechin affects the fat metabolism and is therefore a potential therapy for obesity.  Oolong tea has been shown to have this effect, but you do need to drink a great deal of it.

Oolong tea could decrease body fat content and reduce body weight through improving lipid metabolism. Chronic consumption of oolong tea may prevent against obesity.


ABSTRACT Various health benefits of the cocoa flavanol (-)-epicatechin (EC) have been attributed to its antioxidant and anti-inflammatory potency. In the present study we investigated whether EC is able to prevent deterioration of the anti-inflammatory effect of the glucocorticoid (GC) cortisol in the presence of oxidative stress. It was found that cortisol reduces inflammation in differentiated monocytes. Oxidative stress extinguishes the anti-inflammatory effect of cortisol, leading to cortisol resistance. EC reduces intracellular oxidative stress as well as the development of cortisol resistance. This further deciphers the enigmatic mechanism of EC by which it exerts its anti-inflammatory and antioxidant action. The observed effect of the cocoa flavanol EC will especially be of relevance in pathophysiological conditions with increased oxidative stress and consequential GC resistance and provides a fundament for the rational use of dietary antioxidants

Background: Consumption of flavonoid-rich beverages, including tea and red wine, has been associated with a reduction in coronary events, but the physiological mechanism remains obscure. Cocoa can contain extraordinary concentrations of flavanols, a flavonoid subclass shown to activate nitric oxide synthase in vitro.
Objective: To test the hypothesis that flavanol-rich cocoa induces nitric-oxide-dependent vasodilation in humans.
Design: The study prospectively assessed the effects of Flavanol-rich cocoa, using both time and beverage controls. Participants were blinded to intervention; the endpoint was objective and blinded.
Methods: Pulse wave amplitude was measured on the finger in 27 healthy people with a volume-sensitive validated calibrated plethysmograph, before and after 5 days of consumption of Flavanol-rich cocoa [821 mg of flavanols/day, quantitated as (−)-epicatechin, (+)-catechin, and related procyanidin oligomers]. The specific nitric oxide synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME) was infused intravenously on day 1, before cocoa, and on day 5, after an acute ingestion of cocoa.
Results: Four days of flavanol-rich cocoa induced consistent and striking peripheral vasodilation (P = 0.009). On day 5, pulse wave amplitude exhibited a large additional acute response to cocoa (P = 0.01). l-NAME completely reversed this vasodilation (P = 0.004). In addition, intake of flavanol-rich cocoa augmented the vasodilator response to ischemia. Flavanol-poor cocoa induced much smaller responses (P = 0.005), and none was induced in the time-control study. Flavanol-rich cocoa also amplified the systemic pressor effects of l-NAME (P = 0.005).
Conclusion: In healthy humans, flavanol-rich cocoa induced vasodilation via activation of the nitric oxide system, providing a plausible mechanism for the protection that flavanol-rich foods induce against coronary events.


The Kuna Indians, who reside in an archipelago on the Caribbean Coast of Panama, have very low blood pressure (BP) levels, live longer than other Panamanians, and have a reduced frequency of myocardial infarction, stroke, diabetes mellitus, and cancer—at least on their death certificates. One outstanding feature of their diet includes a very high intake of flavanol-rich cocoa. Flavonoids in cocoa activate nitric oxide synthesis in healthy humans. The possibility that the high flavanol intake protects the Kuna against high BP, ischemic heart disease, stroke, diabetes mellitus, and cancer is sufficiently intriguing and sufficiently important that large, randomized controlled clinical trials should be pursued.

Glutathione reductase (GR) and (partial) Glutathione reductase deficiency

Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) to the sulfhydryl form glutathione (GSH), which is a critical molecule in resisting oxidative stress and maintaining the reducing environment of the cell.

Glutathione reductase reduces one mole of GSSG to two moles of GSH.

Glutathione reductase deficiency is a “rare” disorder in which the glutathione reductase activity is absent from erythrocytes, leukocytes or both. In one study this disorder was observed in only two cases in 15,000 tests for glutathione reductase deficiency performed over the course of 30 years. In the same study, glutathione reductase deficiency was associated with cataracts and favism in one patient and their family, and with severe unconjugated hyperbilirubinemia in another patient. It has been proposed that the glutathione redox system (of which glutathione reductase is apart) is almost exclusively responsible for the protecting of eye lens cells from hydrogen peroxide because these cells are deficient in catalase, the enzyme which catalyzes the breakdown of hydrogen peroxide, and the high rate of cataract incidence in glutathione reductase deficient individuals.

Some patients exhibit deficient levels of glutathione activity as a result of not consuming enough riboflavin in their diets. Riboflavin is a precursor for FAD, whose reduced form donates two electron to the disulfide bond which is present in the oxidized form of glutathione reductase in order to begin the enzyme's catalytic cycle.
In 1999, a study found that 17.8% of males and 22.4% of females examined in Saudi Arabia suffered from low glutathione reductase activity due to riboflavin deficiency.


Glutathione reductase (GR) is a ubiquitous enzyme required for the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) concomitantly oxidizing reduced nicotinamide adenine dinucleotide phosphate (NADPH) in a reaction essential for the stability and integrity of red cells. Mutations in the GR gene and nutritional deficiency of riboflavin, a co-factor required for the normal functioning of GR, can cause GR deficiency. We conducted a study on 1691 Saudi individuals to determine the overall frequency of GR deficiency and to identify whether the deficiency results from genetic or acquired causes or both. The activity of GR was measured in freshly prepared red cell haemolysate in the presence and absence of flavin adenine dinucleotide (FAD) and the activity coefficient (AC) was determined. Samples with low GR activity (> 2.0 IU/g haemoglobin) both in the presence and absence of FAD and an AC between 0.9 and 1.2 were considered GR-deficient. Samples with AC > or = 1.3 were considered riboflavin-deficient. The overall frequency of partial GR deficiency was 24.5% and 20.3% in males and females respectively. In addition, 17.8% of males and 22.4% of females suffered from GR deficiency due to riboflavin deficiency. This could be easily corrected by dietary supplementation with riboflavin. No cases of severe GR deficiency were identified.

Regular readers may recall something very similar with biotin and its enzyme biotinidase.  Biotinidase deficiency is supposedly such a rare metabolic disorder that it is no longer screened for; however, in an autism study in Crete, Greece it was found that partial biotinidase deficiency was quite common.

Glutathione peroxidase

Glutathione peroxidase (GPx) is the general name of an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage.
The biochemical function of glutathione peroxidase is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water.

In earlier posts on anti-oxidants we saw the following presentation from the German scientist.  Note Glutathione (GSH) peroxidases, left halfway down

Glutamate Cysteine Ligase (GCL)

Glutamate Cysteine Ligase (GCL) is the first enzyme of the cellular glutathione (GSH) biosynthetic pathway.

GSH, and by extension GCL, is critical to cell survival.

Nearly every eukaryotic cell, from plants to yeast to humans, expresses a form of the GCL protein for the purpose of synthesizing GSH

Dysregulation of GCL enzymatic function and activity is known to be involved in the vast majority of human diseases, such as diabetes, Parkinson's disease, Alzheimers disease, COPD, HIV/AIDS, and cancer. This typically involves impaired function leading to decreased GSH biosynthesis, reduced cellular antioxidant capacity, and the induction of oxidative stress.

Measuring GR, GPx, GCL in Autism

Fortunately somebody has already measured GR, GPx and GCL in autism, and not surprisingly they are all dysfunctional.  The paper is by the Chauhans, who already feature on my Dean’s list of researchers.

In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (P<0.05). Although there was no significant difference in GR activity between autism and control groups, 40% of autistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired activities in the cerebellum in autism, and lower GCL activity in autism may be related to decreased protein expression of GCLM.

GCLM referred to above is Glutamate-cysteine ligase, it is the first rate limiting enzyme of glutathione synthesis, it is encoded by the GCLM gene. This is an enzyme/ gene you would want to upregulate.

Fortunately we can upregulate GPx enzyme activity with catechin or epicatechin.




The objective of this study was to investigate the effects of catechin and epicatechin on the activity of the endogenous antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) (as well as the total antioxidant capacity (TAC)) of rats after intra-peritoneal (i.p.) administration.


Twenty-four Wistar rats were randomly divided into two groups: the experimental group which was administered daily with a 1:1 mixture of epicatechin and catechin at a concentration of 23 mg/kg body weight for 10 days and the control group which was injected daily with an equal amount of saline. Blood and urine samples were collected before and after the administration period, as well as 10 days after (follow-up).


Intra-peritoneal administration of catechins led to a potent decrease in GPx levels and a significant increase in SOD levels. TAC was significantly increased in plasma and urine. Malonaldehyde levels in urine remained stable. In the animals treated with catechins, SOD activity showed a moderate negative correlation with GPx activity.


Boosting the activity of the antioxidant enzymes could be a potential adjuvant approach for the treatment of the oxidative stress-related diseases.

The objective of this study was to determine whether i.p. administration of catechin and epicatechin could affect the activity of the antioxidant enzymes, SOD and GPx, as well as the TAC in RBCs, blood plasma, and urine.
The antioxidant enzymes are agents that promote reactions for the removal of reactive species (e.g. O2,.H2O2, etc.). They constitute the first line of
defense against oxidative stress. In conditions of increased oxidative stress, the upregulation of the enzyme activity or even, a possible protection of the enzymessubstrate could be of great importance.

Oxidative stress disturbing homeostasis can be resolved by the application of catechins and epigallocatechin gallate (EGCG)18 and there is growing evidence that, the protection, offered by flavonoids and their in vivo metabolites, is not mediated primarily by H-donating antioxidant processes, but is likely to be partly mediated through specific actions, within signaling pathways.

Catechin and epicatechin administration modulated the activity of SOD and GPx but the overall TAC of the RBCs and of the rats plasma remained stable.
Catechins are considered as potent antioxidants and many of their biological actions have been attributed to that. It would have been expected that since catechins are potent antioxidants in vitro, they would have exerted their classical hydrogen-donating antioxidant activity leading to an increase in TAC; as it is seen in the TAC of plasma. The modulation of the enzymes activity may provide evidence that, catechins exert their primary antioxidant activity by specific action within specific molecular pathways, rather than as scavengers of free radicals.

Oxidative stress is a prominent feature of many acute and chronic diseases and even of the normal aging process. The normal function of the antioxidant enzymes guarantees the preservation of cell integrity and thus they can be considered as potential therapeutic targets of oxidative stress-related diseases.
Various antioxidants are available for therapeutic use but most of them have failed in clinical studies of diseases correlated with oxidative stress. Our results suggest that catechins exert their activity not only by H-donating antioxidant processes but likely through mechanisms and pathways that directly or indirectly regulate the expression of the enzymatic antioxidants.

The understanding of these pathways could be important, in developing pharmacological strategies against oxidative stress-related diseases.

For those with autism plus GI issues / ulcerative colitis :- 

Background. This study was pathway of (−)-epicatechin (EC) in the prevention and treatment of intestine inflammation in acute and chronic rat models. Methods. Intestine inflammation was induced in rats using TNBS. The morphological, inflammatory, immunohistochemical, and immunoblotting characteristics of colon samples were examined. The effects of EC were evaluated in an acute model at doses of 5, 10, 25, and 50 mg/kg by gavage for 5 days. The chronic colitis model was induced 1st day, and treated for 21 days. For the colitis relapse model, the induction was repeated on 14th. Results. EC10 and EC50 effectively reduced the lesion size, as assessed macroscopically; and confirmed by microscopy for EC10. The glutathione levels were higher in EC10 group but decreased COX-2 expression and increased cell proliferation (PC) were observed, indicating an anti-inflammatory activity and a proliferation-stimulating effect. In the chronic colitis model, EC10 showed lower macroscopic and microscopic lesion scores and increase in glutathione levels. As in the acute model, a decrease in COX-2 expression and an increase in PC in EC10, the chronic model this increase maybe by the pathway EGF expression. Conclusion. These results confirm the activity of EC as an antioxidant that reduces of the lesion and that has the potential to stimulate tissue healing, indicating useful for preventing and treating intestine inflammation.


We studied a polyphenol-enriched cocoa extract (PCE) with epicatechin, procyanidin B2, catechin, and procyanidin B1 as the major phenolics for its anti-inflammatory properties against dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. PCE reduced colon damage, with significant reductions in both the extent and the severity of the inflammation as well as in crypt damage and leukocyte infiltration in the mucosa. Analysis ex vivo showed clear decreases in the production of nitric oxide, cyclooxygenase-2, pSTAT-3, and pSTAT1α, with NF-κB p65 production being slightly reduced. Moreover, NF-κB activation was reduced in RAW 264.7 cells in vitro. In conclusion, the inhibitory effect of PCE on acute UC induced by DSS in mice was attenuated by oral administration of PCE obtained from cocoa. This effect is principally due to the inhibition of transcription factors STAT1 and STAT3 in intestinal cells, with NF-κB inhibition also being implicated.

 Here is an excellent paper on oxidative stress.  It is about COPD, but applicable to any condition in which oxidative stress is present.


The following paper would suggest that people with COPD would benefit from epicatechin.

The cocoa flavanol (-)-epicatechin protects the cortisol response.


Various health benefits of the cocoa flavanol (-)-epicatechin (EC) have been attributed to its antioxidant and anti-inflammatory potency. In the present study we investigated whether EC is able to prevent deterioration of the anti-inflammatory effect of the glucocorticoid (GC) cortisol in the presence of oxidative stress. It was found that cortisol reduces inflammation in differentiated monocytes. Oxidative stress extinguishes the anti-inflammatory effect of cortisol, leading to cortisol resistance. EC reduces intracellular oxidative stress as well as the development of cortisol resistance. This further deciphers the enigmatic mechanism of EC by which it exerts its anti-inflammatory and antioxidant action. The observed effect of the cocoa flavanol EC will especially be of relevance in pathophysiological conditions with increased oxidative stress and consequential GC resistance and provides a fundament for the rational use of dietary antioxidants.


It would seem that in someone with autism epicatechin is worth a try, other indicators might well include:-

·        Low MOCOS
·        Low ferritin
·        Oxidative stress

And even

·        Restless leg syndrome (symptom of low ferritin)
·        Poor sleep patterns (symptom of low ferritin)

Boosting anti-oxidant enzymes (via gene expression) may be a useful add-on therapy to anti-oxidants themselves.  This is likely true for COPD and autism/schizophrenia.

If you are wondering whether there is anemia or iron deficiency in autism, your questions are likely answered here:-

This research considers the prevalence of iron deficiency in children with autism and Asperger syndrome and examines whether this will influence guidelines and treatment. Retrospective analysis of the full blood count and, as far as available, serum ferritin measurements of 96 children (52 with autism and 44 with Asperger syndrome) was undertaken. Six of the autistic group were shown to have iron deficiency anaemia and, of the 23 autistic children who had serum ferritin measured, 12 were iron deficient. Only two of the Asperger group had iron deficiency anaemia and, of the 22 children who had their serum ferritin measured, only three were iron deficient. Iron deficiency, with or without anaemia, can impair cognition and affect and is associated with developmental slowing in infants and mood changes and poor concentration in children. This study showed a very high prevalence of iron deficiency in children with autism, which could potentially compromise further their communication and behavioural impairments.

As we saw with biotin and soon will with vitamin B12, it seems that people with autism can have unexpected deficiencies of key substances even though their diet may not be deficient.  The identified iron deficiency is an iron storage deficiency.  With biotin the body was unable to recycle the vitamin biotin, due to a problem with the enzyme biotinidase, hence there was a deficiency.

Correcting these deficiencies is quite simple and may well improve any related autism symptoms.  In people without these dysfunctions/deficiencies any such supplements would yield no benefit and might even produce side effects.