On genes, environment, broccoli and autism (again)

Picture: Carl Warner: http://www.carlwarner.com/

I'm serving up two peer-reviewed papers for your reading delight today which draw attention to the ideas that (a) the 'causes' of autism are likely complex and as heterogeneous as the label itself, (b) gene x environment interactions affecting risk of autism are starting to get some good scientific research airtime and (c) don't 'dis the broccoli [chemical] autism connection just yet...

The first paper by Brandon Pearson and colleagues [1] (open-access) has already found some media interest as per the Guardian headline: 'Agricultural fungicides are 'bad news for neurons', study suggests'. Exposing mouse neurons - "cortical neuron-enriched cultures" - to several hundred chemicals (careful of that word) found in the modern environment, researchers concluded that several compounds "produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism, advanced age and neurodegeneration (Alzheimer’s disease and Huntington’s disease)." That is, several types of chemicals quite commonly found in the modern environment seemed to alter gene expression in those mouse neuron enriched cultures that weren't a million miles away from that noted previously in conditions such as autism for example.

Mouse neurons, you might be thinking? Well, obviously one has to be a little cautious about extrapolating from mouse to humans (see here) but researchers did include some comparison analysis looking at "the gene expression profile of our cultures with brain cell-type-specific expression data sets and human brain gene expression data sets." The result: "cortical cultures show strong transcriptional similarities to the human brain."

Clustering chemicals based on "concordant gene expression changes", six groups emerged. Cluster 2 chemicals, containing such pesticides as rotenone, pyridaben and fenpyroximate  and also various compounds under the heading of the strobilurins seemed show some particularly interesting results insofar as they "mimicked the transcriptional changes of two post-mortem ASD [autism spectrum disorder] brain expression data sets in a bidirectional manner." The effects of this cluster of compounds also seemed to unite various conditions with autism including Alzheimer’s disease and Huntington’s disease and the "aging brain". My interest was particularly piqued by that last association in light of other research results (see here).

When it came to the 'effects' of those chemicals in terms of genetic and biological processes, researchers put forward some not unfamiliar potential roles: "These chemicals, most of which inhibit mitochondrial complex I or III, stimulated free radical production and disrupted microtubules." Words like 'oxidative stress' start to emerge as they have done in previous autism research (see here) and yet again, inflammation or inflammatory processes seem also to be indicated. Indeed, the authors also make mention of how effects such as free radical production "can be reduced by pretreating with a microtubule stabilizer, an antioxidant, or with sulforaphane." Yes indeed, sulforaphane - the chemical found in broccoli - might indeed be moving back up the autism research agenda (see here for some previous background).

There is obviously lots more work to do in this area before anyone gets too carried away. The authors note: "While usage and residue levels of cluster 2 chemicals on conventionally grown foods are increasing, in the absence of causality, it is premature to draw correlations with the increased prevalence of ASD and other brain disorders." Lessons could be learned from other blanket suggestions about 'chemicals' and autism (see here) as well as an appreciation for the concept of the the plural autisms (see here). Then there are the practicalities of whether ingesting such compounds on food or in water is the same as direct exposure to cortical neuron-enriched cultures? Or indeed, whether there may be other routes of contact? I might also suggest that further studies should focus on looking for the metabolites of such agents too [2] bearing in mind the concept of statistically significant thresholds...

If you're still here after all that, the second paper I want to talk about is that from Sarah Wong and colleagues [3] that has also received a bit of media attention. The focus this time was on a gene called p53 (see here for some background) and how issues with this gene might be 'over-represented' when it comes to autism following on from other work by some of the same authors [3]. First of all, please don't get too fixated by mention of the words 'cancer gene' when it comes to p53 given it's [protein] tumour suppressing capabilities. As I've discussed before, the risk of cancer does not seem to be elevated any more than the general population risk when it comes to autism (see here). Perhaps of greater relevance to the Wong findings is the idea that p53 has other 'activities' such as that related to oxidative stress (yes, that again) and "DNA repair, bioenergetics and mitochondrial DNA (mtDNA) copy number maintenance."

Based on data from CHARGE (beincharge!), researchers garnered blood samples from 66 children diagnosed with an autism spectrum disorder (ASD) and "race-, gender-, and age-matched typically neurodeveloping children (n = 46)" (authors words not mine). They analysed for mtDNA copy number and deletions and p53 gene copy ratios and found them to be "more common in children with AU [autism] and their fathers." The authors translate their findings as pointing to "a role for deficient DNA repair capacity not driven by paternal age." They also suggest that environment might intersect with genetics in relation to 'severity' scores of autism obtained for their cohort: "gene x environment interaction seems to play a greater role in children with autism with less severe symptoms."

Taken together the Pearson and Wong findings point to some interesting 'associations' potentially relevant to [some] autism. The idea that certain components of the modern-day environment might increase the risk of autism is nothing new but the way that Pearson et al went about studying the possible relationship is. The results from Wong et al suggesting that there might be issues with the gene 'whose role is to suppress cellular damage from environmental stressors' suggests that exposure patterns might not necessarily be where it's all at when looking at compound/chemical X or Y in relation to autism risk. I'm also inclined to direct you to some previous discussion about the caspases and autism (see here) in light of the involvement of p53 with the process of apoptosis (programmed cell death) in mind. As I've mentioned before, the biological mechanisms for how people deal with various xenobiotics needs a lot more investigation in autism research circles (see here); something that might similarly extend to genetic mechanisms too.

Oh, and just in case you think that I'm pushing the either/or of genetic and environment when it comes to autism, I'm not, as words like epigenetics spring to mind and the idea that genomic instability might, for example, have quite a few different dimensions (see here)...

To close, I'm thinking of branching out... football (soccer) pundit perhaps?

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[1] Pearson BL. et al. Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration. Nat Commun. 2016 Mar 31;7:11173.

[2] Domingues VF. et al. Pyrethroid Pesticide Metabolite in Urine and Microelements in Hair of Children Affected by Autism Spectrum Disorders: A Preliminary Investigation. Int. J. Environ. Res. Public Health 2016; 13: 388.

[3] Wong S. et al. Role of p53, Mitochondrial DNA Deletions, and Paternal Age in Autism: A Case-Control Study. Pediatrics. 2016. March 31.

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Pearson, B., Simon, J., McCoy, E., Salazar, G., Fragola, G., & Zylka, M. (2016). Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration Nature Communications, 7 DOI: 10.1038/ncomms11173




Wong, S., Napoli, E., Krakowiak, P., Tassone, F., Hertz-Picciotto, I., & Giulivi, C. (2016). Role of p53, Mitochondrial DNA Deletions, and Paternal Age in Autism: A Case-Control Study PEDIATRICS, 137 (4) DOI: 10.1542/peds.2015-1888

Methylphenidate: a repairer of the 'oxidative balance' in ADHD?

A fairly quick post for you today based on the findings reported by Esra Guney and colleagues [1] who examined whether markers of oxidative stress - an imbalance "between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage" - might be something to look at when it comes to cases of attention-deficit hyperactivity disorder (ADHD).

They concluded that, based on a small-ish sample size, there may be more to see when it comes to oxidative metabolism with ADHD in mind. Their findings are not a million miles away from other work in this area [2] bearing in mind the need for further investigations. I might add that given the quite strong links being put forward between autism and issues with oxidative stress (see here) and the quite consistent overlap between autism and ADHD (see here), future work might need to take quite a broad view of any relationship.

Of particular note to me in the Guney paper was mention of how differences in the oxidative stress index before and after intervention (i.e. medication) in their cohort might offer some new ideas about how certain types of medicines 'work' on cases of ADHD. So: "It was also determined that methylphenidate repairs the oxidative balance by increasing antioxidant defence mechanisms."

Methylphenidate (MPH) (known as Concerta or Ritalin) is a medication of choice for many people diagnosed with ADHD. Although by no means an expert on the whys and wherefores of how MPH works, discussions have always been a little unclear as to how something that looks chemically like an amphetamine (a stimulant) seems to have such a calming effect on some of the characteristics of ADHD. As a nootropic (so-called smart drug) the idea that MPH might work as a performance enhancer offers some clues as to how it might impact on ADHD type symptoms but still curiosity remains on it's important effects.

The idea that MPH might, in amongst its various proposed actions, also impact on processes pertinent to oxidative stress is an interesting one. Animal studies have previously suggested that administration of MPH might affect key compounds related to oxidative stress [3] in particular, related to oxidative defences. That being said, evidence has also been produced to suggest that MPH might do more to induce oxidative stress [4] than to solve any issues, so one has to be a little guarded about making too many sweeping generalisations. That drug dose might also be an important factor is something to take on board too.

Assuming further work is forthcoming to further elucidate any role for MPH in relation to the processes of oxidative stress, some intriguing prospects may lie on the research horizon.

Music: Dream Academy - Life In A Northern Town.

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[1] Guney E. et al. Attention Deficit Hyperactivity Disorder and oxidative stress: A short term follow up study. Psychiatry Res. 2015 Jul 8. pii: S0165-1781(15)00448-5.

[2] Joseph N. et al. Oxidative Stress and ADHD: A Meta-Analysis. J Atten Disord. 2013 Nov 14.

[3] Schmitz F. et al. Chronic methylphenidate administration alters antioxidant defenses and butyrylcholinesterase activity in blood of juvenile rats. Mol Cell Biochem. 2012 Feb;361(1-2):281-8.

[4] Martins MR. et al. Methylphenidate treatment induces oxidative stress in young rat brain. Brain Res. 2006 Mar 17;1078(1):189-97.

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Guney, E., Cetin, F., Alisik, M., Tunca, H., Tas Torun, Y., Iseri, E., Isik Taner, Y., Cayci, B., & Erel, O. (2015). Attention Deficit Hyperactivity Disorder and oxidative stress: A short term follow up study Psychiatry Research DOI: 10.1016/j.psychres.2015.07.003

Coenzyme Q10 and autism

The paper by Frederick Crane and colleagues [1] (open-access here) caught my eye recently and their suggestion that when it comes to autism there may be more research to do on coenzyme Q10. Indeed, the old grey-pinkish matter started grinding into action as to whether there may be a wider research literature on CoQ10 with a focus on autism...

A helping hand? @ Wikipedia 

Coenzyme Q10 otherwise known as ubiquinone, has appeared before on this blog for various reasons (see here and see here). Described as a fat soluble vitamin-like substance, there are apparently a few important steps in the production of CoQ10 implicating one to two old friends - aromatic amino acid friends - involving the "synthesis of the benzoquinone structure" from said aromatic friends (tyrosine or phenylalanine) alongside the "synthesis of the isoprene side chain from acetyl-coenzyme A (CoA) via the mevalonate pathway". It's then a case of marrying the two reaction products together to form something which as the name 'co-enzyme' suggests, is pretty important to quite a few enzymatic reactions.

In terms of the functions of CoQ10, well quite a few of them overlap with areas of interest when it comes to autism. The word 'mitochondria' springs up quite a bit and the important role CoQ10 plays in the production of energy or involvement with ATP (adenosine triphosphate) at least. The antioxidant activity that CoQ10 also seems to possess, at least in it's reduced form - ubiquinol - is also something pretty important. Both mitochondrial function and the concept of oxidative stress have surfaced in the autism research literature in recent years (see here and see here).

The Crane paper presents quite a bit of biochemistry pertinent to "a hypothesis that autism is controlled by a coenzyme Q-dependent redox system in the porin channels". Putting aside my reluctance towards grand, over-arching theories about autism (sorry, the autisms) I'll be honest and tell you that I found the reading pretty heavy going on this topic bearing in mind my very limited knowledge on porin channels and all-things when it comes to transportation in and out of cells. I'm not then going to provide some detailed critique of the author's hypothesis aside from mentioning the paper by Freedenfeld and colleagues [2] on the use "of ribose therapy and NADH therapy on children with autism" (NADH being oxidised by coenzyme Q). NADH is something that Crane et al have talked about in other papers too [3].

The wider autism literature on CoQ10 and autism is best described as limited at the moment. I came across the paper by Gvozdjáková and colleagues [4] (open-access here) talking about results from a preliminary open trial of ubiquinol in a small number of children diagnosed with an autism spectrum disorder (ASD). Following an initial daily dose starting at 50mg of "Liquid liposomal ubiquinol" increased to 100mg daily, authors reported on both behaviour and various biochemical measures including CoQ10 (total) and TBARS "the end product of lipid peroxidation in the body". Bearing in mind the study methodology, the authors reported that a: "Beneficial effect of ubiquinol in children with autism has been demonstrated for the first time". That being said, I note that only the 'CoQ10-TOTAL' biochemical measure came out as significantly affected by ubiquinol supplementation (which is kinda what one would expect) and as someone has already [slightly harshly] noted using PubMed Commons "There is no way in which these observations can be interpreted as a "demonstration" of a "beneficial effect".

I do however believe that there may be more to do from a research point of view when it comes to coenzyme Q10 and autism. Thinking back to the Jim Adams double-blind, placebo-controlled trial looking at a vitamin and mineral supplement for autism (see here) I note that their preparation included CoQ10 (see here) among lots of other things. Granted, lots of other nutrients might have been contributory to the effects they noted, but one cannot, at the moment, rule out any specific effect from CoQ10 either. This is something also mentioned in the new-ish paper from Frye & Rossignol [5]. I'm also minded to mention the various discussions about the use of CoQ10 in relation to cases of chronic fatigue syndrome / myalgic encephalomyelitis (CFS/ME) too (see here) as per papers like the one from Maes and colleagues [6]. Following my mantra about autism seemingly not being protective against any other condition or state, one might further consider additional research as to whether overlap or similar processes might unite at least some autism and at least some CFS/ME or not?

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[1] Crane FL. et al. Plasma membrane coenzyme Q: evidence for a role in autism. Biologics. 2014 May 29;8:199-205.

[2] Freedenfeld SH. et al. Biochemical Effects of Ribose and NADH Therapy in Children with Autism. Autism Insights. 2011; 3: 3-13.

[3] Löw H. et al. Putting together a plasma membrane NADH oxidase: a tale of three laboratories. Int J Biochem Cell Biol. 2012 Nov;44(11):1834-8.

[4] Gvozdjáková A. et al. Ubiquinol improves symptoms in children with autism. Oxid Med Cell Longev. 2014;2014:798957.

[5] Frye RE. & Rossignol D. Treatments for Biomedical Abnormalities Associated with Autism Spectrum Disorder. Front. Pediatr. 2014. doi: 10.3389/fped.2014.00066

[6] Maes M. et al. Coenzyme Q10 deficiency in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is related to fatigue, autonomic and neurocognitive symptoms and is another risk factor explaining the early mortality in ME/CFS due to cardiovascular disorder. Neuro Endocrinol Lett. 2009;30(4):470-6.

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Crane FL, Löw H, Sun I, Navas P, & Gvozdjáková A (2014). Plasma membrane coenzyme Q: evidence for a role in autism. Biologics : targets & therapy, 8, 199-205 PMID: 24920882

Got milk [opioid peptides]?

"Bovine and human casein-derived opioid peptides increased genome-wide DNA methylation in the transcription start site region with a potency order similar to their inhibition of cysteine uptake".

Those were the findings from the paper by Malav Trivedi and colleagues [1] (open-access) including Richard Deth on the authorship team. I was really interested to read this paper having met Malav and Dick a few months back and listened very attentively to some potentially important investigations on-going in their lab. Indeed, such results potentially cast a whole new light on some of my own autism research interests looking at the use of a gluten- and casein-free (GFCF) diet (see here) and more latterly, the reformulation of a great opioid antagonist naltrexone, into a handy cream (see here).

Call me a liar? @ Wikipedia 

A few starting details might be useful:

• Without hopefully just regurgitating what the authors said, the focus of this study was to look at whether "morphine and food-derived opioid-peptides can induce alterations in the cellular redox status, DNA methylation... and transcription process, by altering cysteine uptake in SH-SY5Y cells". As far as I see it, there are 3 important elements to explain, all with an autism slant...
• Opioid peptides - short chains of amino acids with opiate-like activity - have been the topic of quite a bit of discussion when it comes to autism. No-one really disputes the fact that when we eat foods containing gluten and casein, the respective proteins are broken down into peptides, some of which have an affinity to our own opioid (morphine) receptors. The controversy when it comes to autism is the suggestion that said opioid peptides might be part and parcel of the effect noted when some people with autism embark on a diet devoid of gluten and casein - the GFCF diet - and might be detectable in certain biofluids (see here). 
• Redox and oxidative stress also have some research form when it comes to autism (see here) and in particular the quite consistent literature on the antioxidant glutathione being perturbed in quite a few cases of autism (see here). No-one really knows why levels of glutathione (GSH) are coming out so consistently low in relation to autism but one suggestion is that a transporter called EAAT3 involved in the uptake of cysteine, an important building block of glutathione, might be misbehaving. This authorship team together with a few other notable names have talked about this process in relation to autism [2] previously which was the topic of a past post on this blog (see here). 
• DNA methylation is also something talked about quite a bit on this blog. Part of that rising star discipline known as epigenetics, the idea is that adding a methyl group to specific sites on DNA has the ability to modify the expression of certain genes. Hypermethylation is generally taken to mean gene silencing.

And then to the paper itself bearing in mind it is open-access:

• This was a study carried out using "SH-SY5Y and Caco2 cancer cell lines". Said cell lines were subjected to the addition of morphine, bovine [cow] beta-casomorphin 1-7, human beta-casomorphin 1-7 or gliadinomorphin 7 at varying doses. The casomorphins are derived from casein, the milk protein and the gliadinomorphin from the gluten protein.
• Radio-labelled cysteine uptake was monitored in cells and was found to be inhibited by the addition of morphine and the various food-derived opioid peptides. Morphine was most effective at inhibiting cysteine uptake followed by bovine beta-casomorphin 1-7 then human beta-casomorphin 1-7 and finally the opioid peptide derived from gliadin. Interestingly, this effect was "blocked by naltrexone".
• Levels of "metabolic intermediaries involved in transsulfuration and methionine methylation cycle pathways" were also examined. This included looking directly at levels of cysteine and GSH alongside other important compounds in this pathway such as methionine and homocysteine (the big H). Again, changes were noted to these compounds as a function of the addition of morphine and opioid peptides: "consistent with decreased GSH synthesis, decreased activity of methionine synthase, and increased transsulfuration of homocysteine to cystathione". Methionine synthase is again, something already talked about by this authorship group (see here).
• Global DNA methylation status was also examined as a function of morphine and opioid peptide addition to the SH-SY5Y cell line. "A 4 h[our] treatment with morphine or milk-derived opioid peptides... caused a significant shift towards increased methylation (i.e. promoter hypermethylation) in the immediate TSS [transcription start site] region". Morphine was once more most effective at increasing genome-wide methylation followed by bovine and then human beta-casomorphin. Ergo, "opioid-induced changes in cysteine uptake, redox status... are associated with significant genome-wide changes in DNA methylation levels".

I find these results to be absolutely fascinating. Of course, as the authors point out, this was a study of cell lines and hence "need to be replicated in other systems before they can be confidently extrapolated to clinical manifestations". But the possibility that elements of food may have such profound knock-on effects pertinent to the way we 'detox' (yes, it is a real concept) and the expression of some of our genes has endless connotations. I might add that the reported effects of human beta-casomorphin for example, on the processes described may also indicate that not all effects are going to be adverse from an evolutionary perspective.

From the point of view of the use of a GFCF diet in cases of autism, this work potentially adds another string to the various explanations as to why diet may be useful for some on the spectrum. So, outside of other ideas on effect including: lactose intolerance (see here), coeliac disease (see here), non-coeliac gluten sensitivity (see here), altering intestinal hyperpermeability (see here), folate receptor autoantibodies (see here), FPIES (see here), et al, linking gluten and/or casein opioid peptides to something like glutathione production or methylation capacity provides even more research fodder for any interested parties.

Finally(!) there is one small additional point to mention about the Trivedi paper. Tucked in at the end of a sentence in the discussion section are the words: "It should be noted that bovine form of BCM7 [beta-casomorphin 1-7] is only released from cows with the A1 genotype and not A2 genotype cows". I've kinda alluded to the concept that not all mammalian forms of casein are the same in a previous post talking about camel milk and autism (see here). As bizarre as it might sound, there is a growing appreciation that different animals (including humans) might not necessarily produce the same kind of milk [3] and that some types of milk might be advantageous over others. The paper by Barnett and colleagues [4] hinted at one effect - gastrointestinal transit time - when comparing A1 and A2 milks. At the moment however, the science is still a little hazy for suggesting that the differences between the various types of milk might make some more useful for some groups/individuals over others. But certainly there is a potentially intriguing hypothesis requiring some experimental testing there...

Music to close. Paolo Nutini and Pencil Full of Lead.

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[1] Trivedi MS. et al. Food-derived opioid peptides inhibit cysteine uptake with redox and epigenetic consequences. Journal of Nutritional Biochemistry. 2014. June 9.

[2] Waly MI. et al. Prenatal and Postnatal Epigenetic Programming: Implications for GI, Immune, and Neuronal Function in Autism. Autism Res Treat. 2012;2012:190930.

[3] Kamiński S. et al. Polymorphism of bovine beta-casein and its potential effect on human health. J Appl Genet. 2007;48(3):189-98.

[4] Barnett MP. et al. Dietary A1 β-casein affects gastrointestinal transit time, dipeptidyl peptidase-4 activity, and inflammatory status relative to A2 β-casein in Wistar rats. Int J Food Sci Nutr. 2014 Mar 20.

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Trivedi, M., Shah, J., Al-Mughairy, S., Hodgson, N., Simms, B., Trooskens, G., Van Criekinge, W., & Deth, R. (2014). Food-derived opioid peptides inhibit cysteine uptake with redox and epigenetic consequences The Journal of Nutritional Biochemistry DOI: 10.1016/j.jnutbio.2014.05.004

That recent metabolomics study on autism (part 2)

Consider this a part-two to my previous post introducing the paper from Dr Xue Ming and colleagues* on metabolomics, amino acids and gut bacteria with autism in mind. It's going to be another micropost (sort of!) given that quite a bit of the background has already been discussed previously.

So straight to the paper:

• As expected, the study from Yap and colleagues** (including Prof. Nicholson) was the template for undertaking this study.
• Spot urine samples were collected from 48 children diagnosed with DSM-IV autism; in some cases confirmed by the gold-standards that are ADOS and ADI, and compared with 53 matched asymptomatic controls.
• Alongside the various screening measures, a gastrointestinal [disorder] GI severity scale was used to ascertain the presence of GI dysfunction. (verified in 29/48 children with autism).
• Mass spectrometric analysis of the urine sample, blah, blah, blah, and out of a total of 391 metabolites, confirmed metabolites, located in samples, 82 of them were altered between autism and control samples.
• Amino acids et al: several amino acids were significantly lower in the autism group including glycine, serine and the alanines. Taurine also featured. Indeed quite a few of the gamma glutamyl amino acids were also reduced in the autism group with a link to GI disorders potentially suggestive of issues with gamma glutamyl transpeptidase.
• Gut bacterial metabolites: some really interesting stuff here and very much influenced by the presence of GI problems or not. Of particular note, "significantly increased levels of 2-(4-hydroxyphenyl)propionate and taurocholenate sulfate" and reduced levels of 3-(3-hydroxyphenyl)propionate and 5-aminovalerate.
• Mention is also made of some of the gut bacterial findings previously made with autism in mind, and even the possibility of gut hyperpermeability (leaky gut) as influencing the absorption of metabolites. Think back to Paul Patterson' recent announcement on mice, leaky gut and gut bacteria.

A couple of quick observations. Taurine: OK, more an organic acid than an amino acid. Found to be significantly lower in the autism group results compared with controls in this study, but by contrast, elevated in the Yap study. One could argue that there were participant geographical differences (Ming: USA vs. Yap: Australian/Swiss) which might reflect genetic, dietary or environmental differences across different geographical groups. It's interesting to note also that in the Yap paper they noted that "taurine concentrations were hypervariable in the autism group". That and the differences across analytical technologies (Ming: mass spec vs. Yap: NMR). Autisms not autism? Who knows?

Lower urinary glycine and indeed N-acetylglycine were also picked up in the autism group of the Ming study. I don't want to make too much of this at the moment but perhaps will throw in two possibly relevant things: (a) that very interesting paper from Andrew Clayton on autism, aromatic amino acids and gut bacteria (see this post) which talked about benzoic acid and hippuric acid and the link with glycine and (b) the very interesting area of glycine and sleep (see this post). I'm not making any value judgements bearing in mind the focus on urinary glycine not plasma levels of glycine, so just putting it out there.

There's not too much more to say on the the Ming paper apart from being a really interesting piece of research and with a strong requirement for independent scientific replication. That and given the focus on comorbid GI conditions appearing alongside some cases of autism, perhaps a lot more inspection into the root causes and management of them. Indeed look no further than the special Pediatrics supplement on autism for quite an interesting opinion paper*** on this topic with some pretty big hitters as part of the authorship group.

And rest.

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* Ming X. et al. Metabolic perturbance in autism spectrum disorders: a metabolomics study. J Proteome Res. October 2012.

** Yap IK. et al. Urinary metabolic phenotyping differentiates children with autism from their unaffected siblings and age-matched controls. J Proteome Res. 2010; 9: 2996-3004.

*** Coury DL. et al. Gastrointestinal conditions in children with autism spectrum disorder: developing a research agenda. Pediatrics. 2012; 130: S160-S168.

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Ming X, Stein TP, Barnes V, Rhodes N, & Guo L (2012). Metabolic Perturbance in Autism Spectrum Disorders: A Metabolomics Study. Journal of proteome research PMID: 23106572

More metabolomics, amino acids, gut bacteria and autism

"Here he goes again" I hear you say as today's micro-post focuses on a rather interesting paper from Xue Ming and colleagues* which, and I quote, "detected abnormal amino acid metabolism, increased oxidative stress, and altered gut microbiomes in ASD".

Ur-inal or mine? @ Wikipedia  

The name of the game was a favourite -omics of mine, metabolomics (see this recent post for a brief overview) and in particular how the appliance of some really quite powerful science to urine specimens provided by children with autism and aysmptomatic controls, revealed some interesting differences.

A brief overview of the paper and results bearing in mind that I'm still pursuing the full-text paper:

• Using a combination of both liquid- and gas chromatography based mass spectrometry, urine specimens were analysed for 48 children diagnosed with as autism spectrum disorder (ASD) and 53 age-matched not-autism controls.
• Results: after some nifty statistical analysis, urinary amino acid output was one of the most important factors and in particular several amino acids presenting in lower, concentration controlled levels in the ASD group over controls. 
• With shadows of the findings from Prof. Jeremy Nicholson a few years back (see this post), the authors conclude that "several gut bacterial metabolites were significantly altered in ASD children who had gastrointestinal dysfunction".

As indicated, this post is a preliminary one and I hope to fill in a few gaps when I get the full-text paper. Nevertheless, such work as this from Ming and colleagues is indeed important fodder for this blog. We've been here before both with autism and indeed conditions like schizophrenia too albeit not necessarily presenting with the same patterns of findings. Indeed, very much like the rising prominence of disciplines like metabolomics to conditions like schizophrenia - five serum and one urine metabolite for example - there is perhaps some important data to be derived from such studies, at least on the functional differences between autism and not-autism. That outside of the known metabolic disorders with a focus on amino acid chemistry that can present with autism or autism-like characteristics.

The gut bacteria-autism connection? Don't get me started again, as once more another study suggests some potential involvement from those trillions of bacterial masters. I haven't forgotten the old correlation does not equal causation mantra by the way, just pointing out that something seems a little bit unusual with gut bacteria in some cases of autism.

More to follow...

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* Ming X. et al. Metabolic perturbance in autism spectrum disorders: a metabolomics study. J Proteome Res. October 2012.

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Ming X, Stein TP, Barnes V, Rhodes N, & Guo L (2012). Metabolic Perturbance in Autism Spectrum Disorders: A Metabolomics Study. Journal of proteome research PMID: 23106572

Micronutrients for stress and anxiety after earthquake

Lycosthènes @ Wikipedia

The junction between stress, anxiety and environment is of particular interest to this blog. I don't pretend to be an expert on the various mechanisms at work in such a relationship but, based on quite a thorough reading of some of the autism research literature in this area, I am beginning to understand how anxiety, chronic anxiety in particular, is a debilitating condition and plays a primary role in the reduction in quality of life for many, many people.

Moving slightly away from the autism link, I've talked before about some interesting research by Julia Rucklidge and colleagues at the University of Canterbury, New Zealand and their studies following the 2011 earthquake described at the time as " New Zealand's darkest day".

The main premise of that research was that supplementation with various micronutrients - vitamins, minerals and other compounds - may increase 'resiliance' to stress and anxiety associated with highly stressful events like earthquake in individuals diagnosed with ADHD and asymptomatic groups. Quite a bold claim I'm sure you would agree.

Today I'm following up that earlier post as a result of the fairly recent publication of this paper by Dr Rucklidge and colleagues* looking at different micronutrient formulations and their impact on stress and emotions linked to the 2011 earthquake (see trial database entry here). I apologise that I cannot post a link to the full-text paper but hope a short summary from me will suffice:

• Two formulations were evaluated which, to save any commercial advertising issues, I will refer to as B and C, dispensed to 91 participants exhibiting heightened anxiety or stress a few months after the earthquake (B: n =30; C low dose: n=31; C high dose: n=30) based on a random allocation. A separate group of 25 people who either initially declined to participate or did not meet inclusion criteria acted as a non-randomised control group.
• Various measures of anxiety and stress were delivered and completed at baseline and 4-weeks (study end). Measures included: the Depression Anxiety & Stress Scale (DASS), the Traumatic Exposure Severity Scale (TESS) and the Impact of Event Scale (IES) alongside other measures.
• Results: all three treatment groups showed a significant decrease in measures of anxiety and stress following supplementation (based on change in scores between baseline and 4-weeks active treatment). Comparisons with the non-supplemented control group also revealed significant improvements in "stress, anxiety, avoidance, and arousal after 4 weeks of consumption of micronutrients".
• Across the two preparations and different dosages, there were a few differences related to mood, anxiety and energy which have been put down the slightly broader spectrum of nutrients included in formulation C over formulation B. 

Appreciating that there are a number of potential forms of bias to be had in this type of research including a lack of blinding and no placebo as a control, I have to say that I am quite impressed with the reported findings. The authors have gone to some lengths to control for as many factors as possible (being free of psychotropic medication, exclusion of various conditions/states including neurological disorders) and looked at various angles including SES, alcohol/caffeine/smoking/drug use, mental health history, etc as potential influencing variables. Also importantly reporting potential adverse effects from supplementation.

I kinda like the suggestion that the stress and anxiety of being involved in such a horrific natural act might to some degree be abated by simple supplementation with an off-the-shelf micronutrient supplement. Even if it is purely a placebo action - 'take this pill it will help with your anxiety and stress' - giving a daily vitamin-mineral supplement after a disaster like that knocking out infrastructure and interfering with access to food for example, is probably not such a bad idea also in light of the increased risk for disease following such natural disasters (see the Haiti earthquake for a good example). It has to be slightly less expensive and more easy to administer than a few sessions of behavioural therapy or a course of more traditional pharmacotherapy even just as an emergency measure?

The authors do discuss some of the research on how certain components of the micronutrient preparations used might potentially be able to affect symptoms like stress. I'm taken back to the work of David Kennedy and colleagues at Northumbria University and their reports on high-dose vitamin B and vitamin C supplementation on perceived stress in healthy volunteers** (under placebo-controlled, double-blind conditions). There are several hundred more references on this topic also.

Finally, one has to wonder how deep the rabbit hole goes with regards to micronutrient supplementation and stress and anxiety. Outside of the natural disaster bit, could this work imply that nutritional supplementation might be an alternative route to treating anxiety and stress in the general population? (or at least some cases of anxiety and stress). Physiologically what are the mechanisms of effect, acknowledging snippets of research of interest to me on things like gut hyperpermeability and stress (see here)? What about those trillion or so bacteria which reside in our deepest, darkest bowels; how do they respond to stress and what action can they exert? (I note that this same team are going to be looking at a probiotic formulation in a subsequent trial). Even the possibility of trauma being transmitted across generations as per this interesting post on pregnant 9/11 survivors and the emerging field of epigenetics? Lots and lots of questions to answer.

To finish, how about a little piano genius from Ben Folds Five (and Fraggle Rock?).

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* Rucklidge JJ. et al. Shaken but unstirred? Effects of micronutrients on stress and trauma after an earthquake: RCT evidence comparing formulas and doses. Human Psychopharmacology. July 2012.

** Kennedy DO. et al. Effects of high-dose B vitamin complex with vitamin C and minerals on subjective mood and performance in healthy males. Psychopharmacology. 2010; 211: 55-68.

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Rucklidge JJ, Andridge R, Gorman B, Blampied N, Gordon H, & Boggis A (2012). Shaken but unstirred? Effects of micronutrients on stress and trauma after an earthquake: RCT evidence comparing formulas and doses. Human psychopharmacology PMID: 22782571

Brain glutathione redox status, Brussels sprouts and autism?

Sprouts, sprouts, sprouts @ Wikipedia

Glutathione in relation to autism spectrum conditions is a bit of a favourite topic of mine. I've talked about glutathione a few times on this blog (here and here) and how the various reports on a reduced level of functioning of this fantastic part of our antioxidant system seem to show more than a passing connection to cases of autism. That and the fact that consumption of a favourite foodstuff of mine, the Brussels sprout, might have quite a positive influence on some of the inner workings of the glutathione system (see Nijhoff and colleagues*) makes it a compound of some interest.

Up until this point, the collected research looking at glutathione in relation to autism had however tended to be focused on circulating levels of glutathione in its various forms alongside the enzymes supporting its important tasks. Enter then a paper published by Rose and colleagues* (full-text) on glutathione, the brain and autism previously described at IMFAR 2012 (here). You may have spotted a few familiar names on the authorship panel of this paper including Jill James (hypomethylation and autism) and Richard Frye (folate receptor autoantibodies).

Aside from a couple of forays into the world of brain research and the autism spectrum (see here for example), I have tended to keep away from discussing such investigations in too much detail on this blog. My reasoning: adhering to the phrase 'a cobbler should stick to his last', coupled with a view that the various research focused on the brain and autism just seemed so darned complicated. Suffice to say that it all brings back blurred memories of my undergraduate days where I admit to being more than a little confused about what brain region was supposed to do what.

In this post I am going to include some discussion on the findings reported by Rose et al albeit with the caveat that my brain may not be up to exploring all the avenues of potential interest related to these findings and what they mean.

Since I am discussing glutathione, I also want to bring to your attention some slightly more 'preliminary' findings reported in this poster by Cruikshank and Wood** on urinary glutathione in relation to autism. The caveat here being that this is not a peer-reviewed piece of research and hence still requires quite a lot more work before being taken as Gospel (despite the recent media interest).

Back to Dr Rose's paper:

• This was a study of post-mortem brain specimens. In light of the recent news of a freezer malfunction linked to the destruction of a number of stored brain tissue samples from people with autism, there is a degree of poignancy to this study reiterating how valuable these types of tissue are to furthering autism research.
• Samples from two areas of the brain - the cerebellum and Brodmann area 22 (BA22) (part of the superior temporal gyrus) - were studied, comparing samples from people with autism (n=15 & n=12 for the two areas) with control specimens. These brain areas have been talked about before with autism in mind (here and here) as the name Eric Courchesne drifts into my consciousness.
• Levels of various compounds were examined in samples including: reduced glutathione (GSH), oxidised glutathione disulfide (GSSG), 3-nitrotyrosine (3-NT) and 3-chlorotyrosine (3-CT); calculating glutathione redox/antioxidant capacity (GSH/GSSG), oxidative protein damage and oxidative DNA damage (8-oxo-deoxyguanosine; 8-oxo-dG). Aconitase activity was also measured.
• The results: in both brain regions, all studied compounds were altered at a group level in cases of autism compared to controls. So, levels of glutathione (GSH) were decreased in both brain areas compared to control samples (43% and 32% reductions in cerebellum and BA22 respectively). Levels of oxidised glutathione disulfide (GSSG) were elevated in autism vs. controls in both brain areas (18% vs. 19% elevations respectively) and overall glutathione redox/antioxidant capacity (GSH/GSSG) was significantly different in autism vs. controls. These findings are roughly in line with what has been reported in studies of other tissues in cases of autism.
• Oxidative stress and oxidative protein damage markers were also significantly elevated in the autism group vs. controls in both brain areas. Aconitase activity was significantly lower in the autism group in the cerebellum but escaped significance in relation to BA22. 
• The authors note: "decreased glutathione-mediated redox/antioxidant capacity previously observed in plasma and immune cells from children with autism is also significantly decreased in two brain regions previously shown to be affected in autism, the cerebellum and BA22".

There is quite a bit of information to take in from this study and as a result, several important things which will require some external replication with suitable age and sex matched controls. Oxidative stress / damage is something that tends to get banded around quite a lot in these days of the free radical. Rose and colleagues have now provided some very important preliminary flesh on the bones to this story, suggesting "functional consequences" on specific brain areas previously linked to autism following their results. Their findings also pretty much confirm what quite a few others have been saying about glutathione in relation to cases of autism: whether causative or epiphenomenal, there's something amiss with the whole oxidative stress / antioxidant balance in at least a proportion of cases of autism and it may well extend beyond just a casual relationship. 


I note also their findings with regards to glutathione redox/antioxidant capacity (GSH/GSSG) and 8-oxo-dG in the cerebellum. Combining autism cases and control data, the authors report on an important relationship between how well the GSH/GSSH capacity performs and the amount of oxidative DNA damage potentially present. Similar things have been reported in other studies so no real surprises there.


I'm not going to get too far into the findings with regards to decreased aconitase activity in the autism cases and its link to mitochondrial oxidative stress. If you really want some reading on the subject, this paper by Cantu and colleagues**** (full-text) should keep you going for a while. Suffice to say that mitochondrial aconitase inactivation might have some pretty negative implications as per this article by Vasquez-Vivar and colleagues***** (full-text).


Just a couple of more things to add then I'm done. The suggestion of "a chronic neuroinflammatory state" at the brain sites under investigation has to be included in any synopsis. Neuroinflammation in autism is a topic that has cropped up quite a few times in the research literature. To pick out one study that springs to mind, the findings from Vargas and colleagues****** and their introduction of microglia into the mix is a case in point. Microglia is another area not readily touched upon this blog so in this case I will perhaps refer you to a nice blog post by Paul Patterson on the topic of hungry microglia potentially eating synapses in autism. I'm not necessarily saying that this is an essential part of the Rose findings but it could be a potentially important tie-in.


The link between elevated levels of 3-NT and elevated nitric oxide (NO) production described in the Rose paper is a final point. The mechanism for this relationship is explained pretty well here. I've covered some of the work on elevated NO metabolites and autism in a previous post. The net results seeming to indicate that levels of NO metabolites are elevated quite consistently in cases of autism. And then there is the inflammation link which has been covered quite a bit before.


OK that's enough for now. You might have realised that I am fairly interested in this paper by Rose and colleagues despite the preliminary nature of their study and the limitations of my knowledge in the area of the brain in autism. Yet another good reason why glutathione in relation to autism deserves a lot more research interest alongside tentative suggestions on whether we might actually be able to do something about it (see previous post). 


To finish, how very dare they make this song which implies a world without Brussels sprouts. You will eat your phenylthiocarbamide and like it...

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* Nijhoff WA. et al. Effects of consumption of Brussels sprouts on intestinal and lymphocytic glutathione S-transferases in humans. Carcinogenesis. 1995; 16: 2125-2128.

** Rose S. et al. Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain. Translational Psychiatry. July 2012.
DOI: 10.1038/tp.2012.61

*** Cruikshank C. & Wood T. Quantitation of glutathione as a urinary autism biomarker (poster).

**** Cantu D. et al. Oxidative inactivation of mitochondrial aconitase results in iron and H2O2-mediated neurotoxicity in rat primary mesencephalic cultures. PLoS ONE. 2009; 4: e7095.

***** Vasquez-Vivar J. et al. Mitochondrial aconitase is a source of hydroxyl radical. The Journal of Biological Chemistry. 2000; 275: 14046-14069.

****** Vargas DL. et al. Neuroglial activation and neuroinflammation in the brain of patients with autism. Annals of Neurology. 2005; 57: 67-81.

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Rose S, Melnyk S, Pavliv O, Bai S, Nick TG, Frye RE, & James SJ (2012). Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain. Translational Psychiatry, 2 PMID: 22781167