Showing posts with label iron deficiency. Show all posts
Showing posts with label iron deficiency. Show all posts

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.