Another blood test for autism?

"These results form the foundation for the development of a biochemical test for ASD [autism spectrum disorder] which promises to aid diagnosis of ASD and provide biochemical understanding of the disease, applicable to at least a subset of the ASD population."

OK, use of the word 'disease' in the context of autism is really, really not OK in this day and age. Researchers, peer reviewers and their publishing journals should be doing something about this kind of language. There are however some potentially important aspects to the work published by Daniel Howsmon and colleagues [1] worth talking about. Not least is their observation on how "folate‐dependent one carbon metabolism (FOCM) and transsulfuration (TS) pathways" that have been quite readily *associated* with autism might be linked to quite a bit more than just uncovering the biochemistry of at least some autism (see here for example).

Before progressing further into these findings, I note there has already been some media interest in them (see here) with a byline reading: "First physiological test for autism proves high accuracy in second trial." We'll see about that...

So, after quite a long introduction about 'biomarkers for autism' and how they "come with their own set of challenges before they reach clinical translation", authors report further results building on some of their previous work in this area [2] that I've already covered on this blog (see here). On that previous research occasion, the suggestion was that between 5 and 7 metabolites linked to folate and/or transsulfuration pathways provided a 'best fit' when it came to picking out children diagnosed with autism from those not diagnosed with autism.

This time around, there was an 'extension' to that work: "(a) By comparing univariate analysis with four different multivariate methods on FOCM/TS data for ASD biomarker development to ensure that the identified results are not restricted to FDA [Fisher Discriminant Analysis] and (b) to test and validate multivariate FOCM/TS biomarkers on data collected from a new cohort of ASD participants." The words 'training data' and 'validation data' are used quite a bit throughout the Howsmon article, illustrating how different statistical classification methods were initially applied to training data from the cohort used in their first paper, which were then tested on a new cohort of participants (n=154) diagnosed with an ASD. Given some of the names included on the authorship list, it's no surprise that participant data with regards to the metabolites being looked at were drawn from other studies looking at the possible clinical value of preparations like folinic acid (see here) and sapropterin (see here) with autism in mind.

When those different statistical classification methods were applied and data was crunched, a few observations were made. The headline result was that one model/method produced the best 'potential' biomarker results and it was the same/similar method to that previously discussed by the authors. To quote: "An FDA model using five variables was shown to slightly outperform the other models on this new validation data set." That being said, the accuracy rates (including false positive and false negative rates) hovering around the high 80%s have to take into account that two of the metabolites thought to be important on the last research occasion - % DNA methylation and 8‐OHG - "were not present in the validation set" on this research occasion. This is a pity and a weakness of the current study.

So, do we at last have a 'physiological test' with 'high accuracy' for picking out autism from not-autism? Erm, not quite yet. With all due respect to the authors, their data is interesting and does partially back up their original findings, but we're not quite there yet with regards to rolling out any sort of biological test for autism. Indeed, in these days of the plural 'autisms' (see here) and acknowledging that the diagnosis of autism rarely presents in some sort of diagnostic vacuum (see here) it could be worthwhile re-evaluating whether we're ever likely to see a 'one biological test to diagnose them all' situation.

Further investigations are however indicated and of course, this more recent information does add to the quite rich data already generated suggesting that quite a bit more focus on things like methionine, homocysteine, cysteine and glutathione in relation to autism could be an important research path to follow. I'm also minded to suggest that different research teams taking on a 'possible biomarker for autism' type research perhaps need to talk more to each other (see here) pooling findings, resources and perhaps participant groups too...

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[1] Howsmon DP. et al. Multivariate techniques enable a biochemical classification of children with autism spectrum disorder versus typically‐developing peers: A comparison and validation study. Bioengineering & Translational Medicine. 2018. May 14.

[2] Howsmon DP. et al. Classification and adaptive behavior prediction of children with autism spectrum disorder based upon multivariate data analysis of markers of oxidative stress and DNA methylation. PLoS Comput Biol. 2017 Mar 16;13(3):e1005385.

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Folate-dependent one carbon metabolism and transsulfuration pathways: biomarkers for autism?

I found it a little unusual that the findings reported by Daniel Howsmon and colleagues [1] (open-access) talking about "multivariate statistical analysis presented herein [provided] unprecedented quantitative classification results for separating participants into ASD [autism spectrum disorder] and NEU [neurotypical] cohorts based solely on biochemical data" merited a rapid reply in a prominent science magazine pouring cold water on the results (see here). Not least because one of the commentators interviewed in said science magazine article is also not seemingly immune when it comes to sweeping claims being made on the basis of preliminary research findings about autism (see here as per the previous headline: 'Super-parenting' improves children's autism).

No mind, the Howsmon paper - including a notable research name on the authorship list - mentions a few important compounds and biological processes in their discussions on: "Stepping towards this goal of incorporating biochemical data into ASD diagnosis." The sorts of things covered included various biological 'markers' pertinent to folate-dependent one-carbon metabolism (FOCM) and transsulfuration (TS) some of which have been fodder for this blog previously (see here and see here for examples). Researchers looked at these various compounds in blood samples from some 80 children diagnosed with an ASD and compared levels with 47 siblings and 76 age-matched controls. They applied some nifty statistics to try and determine whether any combination of the 24 analytes examined might be potential biomarker-material for an autism diagnosis. You'll note that once again the quite problematic binary description of 'neurotypical' was used to define 'not-autism' leading onwards to the inevitable questions: 'what is neurotypical?' and 'what are the boundaries of being neurotypical?' Sensible [evidence-based] answers on a postcard please.

Results: "FDA [Fisher Discriminant Analysis] on seven metabolites allows sufficient separation such that a linear classifier can correctly resolve 96.9% of participants." But actually this was not the whole story as the authors also report that five compounds/variables - GSSG, tGSH/GSSG, Nitrotyrosine, Tyrosine, and fCysteine - provided the best 'fit' when it came to potentially picking out children with autism. You might note that some of those 'famous five' have some autism research history (see here). The authors similarly note that: "these variables are affected by high quality vitamin supplementation that also decreases ASD severity in at least a subset of cases." Mmm.

There is definitely more science to do in this area. Biomarkers in relation to autism have come and gone down the years (see here for example) and I'm not altogether sure that using the label 'autism' as a starting point for this kind of research is necessarily the best idea (see here). Outside of just the heterogeneity and plurality - the autisms - associated with the label autism, there are other considerations to take on board such as the impact of all that over-represented comorbidity too (something that continues to 'mess around' with various 'autism is linked to..' studies).

But that shouldn't stop further efforts in this area including those also looking to expand into the 'genetics' of folate metabolism alongside the biochemistry, as per everyone's favourite scrabble word 'MTHFR' (see here) and its [meta-analysed] potential contribution to some autism. I agree that we are not quite there when it comes to folate metabolism as providing a generic biomarker or set of biomarkers for autism, but there again, the authors never said that it definitively did: "it should be noted that these studies should be replicated and empirically tested on a wider scale before more definite conclusions can be drawn." Too true but the Howsmon results represent an interesting first attempt...

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[1] Howsmon DP. et al. Classification and adaptive behavior prediction of children with autism spectrum disorder based upon multivariate data analysis of markers of oxidative stress and DNA methylation. PLoS Comput Biol. 2017 Mar 16;13(3):e1005385.

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Howsmon DP, Kruger U, Melnyk S, James SJ, & Hahn J (2017). Classification and adaptive behavior prediction of children with autism spectrum disorder based upon multivariate data analysis of markers of oxidative stress and DNA methylation. PLoS computational biology, 13 (3) PMID: 28301476

Decreased brain levels of vitamin B12 in autism

I have to thank Dr Malav Trivedi for bringing my attention to some recent findings reported by Yiting Zhang and colleagues (including Malav) [1] (open-access) suggesting that: "levels of vitamin B12, especially its MeCbl [methylcobalamin] form, decrease with age in frontal cortex of control human subjects."

Further, researchers reported: "abnormally lower total Cbl [cobalamin] and MeCbl levels in subjects with autism and schizophrenia, as compared to age-matched controls." Some media on the findings can also be read here.

Working from the lab of Dr Richard Deth (quite a familiar name to this blog), researchers initially analysed a most precious sample medium (postmortem brain samples) obtained from various biobanks and including various patient groups. So alongside samples from 12 children with autism were samples from 9 people diagnosed with schizophrenia and some 43 'controls' with ages ranging between 19 weeks old and 80 years old. "Changes in Cbl species were compared with the status of methylation and antioxidant pathway metabolites" accompanied by data derived from a knock-out mouse model: "the influence of decreased GSH [glutathione] production on brain Cbl levels was evaluated in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice in which GSH synthesis was impaired, leading to a brain GSH level decrease of 60–70%."

Looking at postmortem frontal cortex brain samples, researchers reported that finding on levels of vitamin B12 - particularly the MeCbl vitamer -  decreasing with age. Bearing in mind the relatively small participant numbers included, the idea that lower brain tissue levels of total cobalamin and methylcobalamin were also present (almost unanimously) in the autism and schizophrenia groups could be important. I might at this point direct readers to previous discussions on vitamin B12 and autism on this blog (see here) including the research idea of supplementing (see here) with no medical advice given or intended.

There are a few other details worth pointing out from the Zhang findings. Analysis of thiols in brain samples across the autism vs control group revealed some potentially interesting data. So, methionine levels were quite a bit lower in the autism group [significantly lower] as were levels of "the methyl donor S-adenosylmethionine (SAM)." Both these compounds form an important part of the whole 'methylation of DNA' process (see here) among other things.

Glutathione, a compound that has seen its fair share of speculation with autism in mind (see here), was also on the research menu in the Zhang study. Interestingly and again bearing mind the small participant numbers, brain levels of this stuff were lower in the autism group as a whole but not significantly so when compared to controls. This finding might map on to other brain studies with autism in mind (see here). Likewise, cysteine (another potentially relevant compound to some autism) produced a similar finding.

I would encourage readers to take some time looking at the Zhang paper. In conjunction with other results reporting on some important elements to the emerging story (see here) I believe there are further studies to be done applicable to the notion that: "impaired methylation may be a critical pathological component" for at least some autism (see here). Indeed, other research papers have also discussed this issue [2]. The idea that studies about human ageing may likewise be informative to autism (and schizophrenia) research also carries quite a lot of traction too.

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[1] Zhang Y. et al. Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PLoS One. 2016 Jan 22;11(1):e0146797.

[2] Keil KP. & Lein PJ. DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders? Environmental Epigenetics. 2016; 1-15.

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Zhang Y, Hodgson NW, Trivedi MS, Abdolmaleky HM, Fournier M, Cuenod M, Do KQ, & Deth RC (2016). Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PloS one, 11 (1) PMID: 26799654

Homocysteine and autism: yet more...

It's been a while since I've discussed the issue of homocysteine - that's homocysteine not homocystine - with autism in mind, so consider this short blog entry a bit of an update to previous discussions (see here and see here).

In case you need to know it, homocysteine is an important component of the trans-sulfuration pathway intersecting with both the methione cycle and the folate cycle. Collectively, these biological processes have important functions for various aspects of biology including the process of methylation and the issue of oxidative stress leading into the important role of glutathione (see here) for example.

I was brought to this post following the publication of two recent paper from Carmen Puig-Alcaraz and colleagues [1] and from Yu Han and colleagues [2] that both reported on elevated levels of homocysteine to be present in their cohorts of children diagnosed with an autism spectrum disorder (ASD) compared with asymptomatic controls. These findings are pretty much in line with what most other research has reported in this area.

Puig-Alcaraz et al reported that alongside an overall increased level of urinary homocysteine in their cohort, there seemed to be something of a relationship between elevated urinary homocysteine with "the severity of the deficit in communication skills" in their participant group. Something that was not seen when looking at the other core areas of autism (social interaction and repetitive/restricted behaviour). I'm intrigued at the prospect that specific traits may be linked to something like elevated levels of homocysteine although recognise the need for far greater scrutiny of this finding with larger cohorts. That this group only measured homocysteine in urine is another issue that needs to be further explored.

Han et al report results based on Chinese children. This in itself is an important cohort suggesting that issues with homocysteine might cross geography and ethnicity when it comes to autism on the basis of other studies looking at different populations. Alongside reporting on elevations in homocysteine, researchers also noted that total levels of glutathione and cysteine were lower in the autism group; findings that accord with meta-analyses of glutathione and related compounds with autism in mind (see here). Further: "Hcy [homocysteine] levels correlated significantly with increasing CARS [Childhood Autism Rating Scale] scores and GSSG [oxidized glutathione] levels in children with ASD" with the proviso that further investigations are needed in this area.

The peer-reviewed evidence is indeed stacking up for something potentially fundamental at work when it comes to autism and homocysteine. At this stage it would be difficult to tease apart homocysteine alone as being 'linked' to [some] autism given the myriad of other compounds/pathways also potentially implicated as per the literature on the B vitamins and their important links to homocysteine. I'm also wondering whether another relation of one of the B vitamins, methylmalonic acid, that has languished in the autism science desert for far too long might also need resurrecting in future studies on the 'big H' and autism?

And yes, screening is important [3]...

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[1] Puig-Alcaraz C. et al. Increased homocysteine levels correlate with the communication deficit in children with autism spectrum disorder. Psychiatry Res. 2015 May 29. pii: S0165-1781(15)00290-5.

[2] Han Y. et al. Abnormal transsulfuration metabolism and reduced antioxidant capacity in Chinese children with autism spectrum disorders. International Journal of Developmental Neuroscience. 2015. July 3.

[3] Ranjan S. & Nasser JA. Nutritional Status of Individuals with Autism Spectrum Disorders: Do We Know Enough? Adv Nutr. 2015 Jul 15;6(4):397-407.

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Puig-Alcaraz C, Fuentes-Albero M, Calderón J, Garrote D, & Cauli O (2015). Increased homocysteine levels correlate with the communication deficit in children with autism spectrum disorder. Psychiatry research PMID: 26070768





Han, Y., Xi, Q., Dai, W., Yang, S., Gao, L., Su, Y., & Zhang, X. (2015). Abnormal transsulfuration metabolism and reduced antioxidant capacity in Chinese children with autism spectrum disorders International Journal of Developmental Neuroscience DOI: 10.1016/j.ijdevneu.2015.06.006

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

Methylcobalamin and folinic acid for autism? Hold it right there...

The title of this post should also have included the word 'glutathione' too based on the results reported by Richard Frye and colleagues* (open-access) describing behavioural and biochemical data from a 3-month open trial of methylcobalamin, a vitamin B12 'vitamer', and folinic acid with a group of children diagnosed with an autism spectrum disorder (ASD).

Dr Frye and his various research are no stranger to this blog; ranging from mitochondrial dysfunction linked to cases of autism (see here), through to tetrahydrobiopterin (BH4) and autism (see here), and folate receptor autoantibodies and autism (see here). Indeed that last link on folate receptor autoantibodies brings into view a potential reason why folinic acid was included in their recent study. That and the inclusion of Jill James on the authorship list and her previous studies looking at combined methylcobalamin and folinic acid supplementation for autism** (open-access) discussed as part of a previous post (see here).

The crux of this study was the suggestion that in the great and complex pathway which links the recycling of homocysteine (the big 'H') and the folate cycle, there is potentially enough going on in cases of autism to interfere with (a) the process of methylation (see here) and (b) that most useful of compounds, glutathione (see here), well reduced glutathione anyway. I'm also inclined to point readers the way of the very thorough analysis of glutathione and autism produced by Main and colleagues*** (open-access) a while back (see here) just in case you think I'm talking biochemical mumbo-jumbo.

Readers might already have seen mention of the words 'open trial' at the top of this post. This indicating that the latest study from Frye and colleagues was a case of following 37 children who fitted the entrance criteria - including "abnormal methylation capacity (SAM/SAH < 3.0) and glutathione redox metabolism (GSH/GSSG < 6.0)" - and seeing how they went over the course of a "sterile subcutaneous injection of methylcobalamin in the fatty tissue of the buttocks" every 3 days combined with oral delivery of folinic acid twice daily mixed with food. For those wincing or furrowing their brows about those injections of methylcobalamin with children with autism, I'll just say that the issue of drug delivery has been talked about in a previous post and this study was passed through an ethics committee "at the University of Arkansas for Medical Sciences".

The results are interesting. Quite a few behavioural changes were documented according to use of the VABS. This bearing in mind that (a) there was no control or placebo group and (b) VABS is a parent-report schedule which in this case merely looked at unblinded pre- and post-intervention scores. Nonetheless, the intervention resulted in "significant increases in VABS scores for all domains, including daily living, social, and communication skills, with an average effect size of 0.59, which is in the medium-to-large range." The authors even went as far to say that the VABS changes indicated something like an average 7.7 month gain over the 3 month period of study.

All well and good with that open-trial caveat well and truly in place. It is however the details regarding the biochemical measure of glutathione measurement that I was more interested in. Indeed, if I had to suggest one improvement to this paper, it would have been to include a simple table showing glutathione measures - GSH/GSSG - at baseline compared with at 3 months. Instead, the glutathione results are all bundled up with the VABS results as per the example of figure 3 showing: "the change in the glutathione redox status (reduced-to-oxidized glutathione ratio) and change in subscales of the Vineland Adaptive Behavior Scale (VABS) subscales".

What I did manage to glean is that: "the overall glutathione redox status was not related to VABS subscales, indicating that overall development did not appear to be related to overall glutathione redox status". Fair enough, a possible selection issue based on the group eventually included for study. It was the change in glutathione redox status after intervention which seemed to tie into the VABS results reported. Still, I would have liked to have the biochemical data presented as a stand-alone table.

I'm trying not to be overly-critical of this paper and results contained within. As with many other researchers, I'm guilty of the odd open-trial forming part of my CV (see here). Whilst useful as a starting point for looking at a particular intervention or trying to get others to do a more methodologically-sound study, one has to be quite cautious of such work and the myriad of biases that they contain.

I do get the impression that outside of just a more methodologically-sound trial, a lot more questions need to be asked about this intervention regime before it can be considered as something more mainstream. Outside of the 2 children who dropped out of the study because "parents were uncomfortable giving the methylcobalamin injections", there's also a question of what such an intervention is actually doing. I note the authors when discussing the previous James trial**, are quoted as saying: "The fact that the treatment [methylcobalamin and folinic acid] improved but did not normalize methionine, SAM and glutathione concentrations may reflect ongoing metabolic compensation for incompletely resolved oxidative stress". This may very well be true, but could also indicate that intervention was also working on other biological systems too.

That also mention is made of the Hardan trial of N-acetlycysteine (NAC) for autism (see here) and NAC being a direct glutathione precursor, suggests to me that when it comes to glutathione production, the shortest point might be A to B bearing in mind what results have been obtained from direct glutathione supplementation**** (open-access).

To close, the Clash have a question for you.... (it's the indecisions which bug me).

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* Frye RE. et al. Effectiveness of Methylcobalamin and Folinic Acid Treatment on Adaptive Behavior in Children with Autistic Disorder Is Related to Glutathione Redox Status. Autism Res Treat. 2013: 609705.

** James SJ. et al. Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism. Am J Clin Nutr. 2009 January; 89(1): 425–430.

*** Main PA. et al. The potential role of the antioxidant and detoxification properties of glutathione in autism spectrum disorders: a systematic review and meta-analysis. Nutr Metab (Lond). 2012 Apr 24;9:35.

**** Kern JK. et al. A clinical trial of glutathione supplementation in autism spectrum disorders. Med Sci Monit. 2011 Dec;17(12):CR677-82.

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Richard Frye, Stepan Melnyk, George Fuchs, Tyra Reid, Stefanie Jernigan, Oleksandra Pavliv, Amanda Hubanks, David W. Gaylor, Laura Walters, S. Jill James (2013). Effectiveness of Methylcobalamin and Folinic Acid Treatment on Adaptive Behavior in Children with Autistic Disorder Is Related to Glutathione Redox Status Autism Research and Treatment DOI: 10.1155/2013/609705

NAC for autism: a case study

NAC or N-acetlycysteine has appeared a couple of times on this blog in relation to both autism (see here) and schizophrenia (see here). Not bad for a compound which more readily finds a home in modern medicine following paracetamol (acetaminophen) overdose or as a consequence of its mucolytic properties.

As one might imagine, the autism link is of particular interest to this blog, focused specifically on the findings of Hardan and colleagues* when it came to putting NAC to the [albeit preliminary] experimental test. The results by the way were encouraging for at least some parts of the presentation of childhood autism with the promise of more to come.

As a sort of follow-up to the Hardan paper, I'm talking today about a case report offered by Ghanizadeh & Derakhshan** (open-access) highlighting a little more individual detail following the use of NAC with an 8-year old boy diagnosed with autism. I know the word 'case report' sends a shudder down many a scientific shoulder, but as I've said quite a few times before, we ignore the N=1 in autism at our peril given the wide, wide heterogeneity present and all that associated comorbidity to contend with. Real personalised medicine you might say.

If I have managed to persuade you to listen to the rest of my ramblings on this paper and topic, there are a few important points to make about/from the Ghanizadeh paper:

• From the description provided, the child in question seemed quite floridly autistic with the important add-ons of hyperactivity and inattention present from an early age. Although we aren't told what exactly it means, the authors note: "His laboratory examination was unremarkable".
• Oral NAC (800mg per day) was begun as part of another trial by the authors to counteract nail-biting***. As unusual as it might sound, mail-biting has been a focus of some NAC research coincident to the presence of anxiety.
• Indeed, the boy's nail-biting behaviours did seem to subside alongside the installation of NAC but perhaps of greater interest were the reports that "there was a marked reduction in his autism symptoms 30 days after the onset of NAC administration". OK so this report did come from the boy's parents, and without causing any offence, the issue of objectivity might come into play.
• The types of 'changes' reported however were in core areas such as his verbal skills, social interaction and a quite unusual preoccupation with having his hair cut (I say unusual because a visit to the barber or hairdresser described by many parents about their child with autism, is often characterised by entirely the opposite reaction).
• Aside from "a mild abdominal pain" the authors importantly say that "nothing worsened after the administration of NAC" which I take to indicate that side effects were minimal over the course of the intervention.

I should have perhaps mentioned at the beginning that there is some sound logic why NAC might have some effect on cases of autism. The amino acid cysteine as well as containing sulfur, so potentially tied into to that most forgotten areas of autism research sulfur chemistry (see here), is also the precursor to another important compound, glutathione. I know my regular readers are probably getting a little bored of me going on about this 'elephant in the room' and in particular that glutathione overview paper by Main and colleagues (see here) but a possible link is a possible link.

I was also interested to read the authors' discussions on how NAC might also have the ability to decrease "high glutamate levels". As any good biochemist will tell you, glutathione, which is dependent on cysteine, is a tripeptide which also incorporates the amino acids glycine and glutamate into its triadic manufacture. From that point of view, circumstances where any of the three amino acids were low or not optimally biologically available might affect the production of glutathione. If that happens to mean you have low cysteine levels, glutathione would be low but also this might mean levels of glutamate or glycine could be higher as a result of not being used up to make glutathione. Glutamate is another compound finding some significant interest with regards to autism and conditions presenting with autistic symptoms.

Reiterating that the Ghanizadeh paper is a case report, I do find there to be some interesting observations reported. With my speculating hat on, I do wonder whether that link with nail-biting and onwards anxiety suggested for NAC might also be part and parcel of the effect observed in this case given the quite considerable link suggested between autism and anxiety (see here)?

And by the looks of things NAC is in the research ascendancy perhaps even with a prophylactic effect****...

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* Hardan AY. et al. A randomized controlled pilot trial of oral N-acetylcysteine in children with autism. Biol Psychiatry. 2012 Jun 1;71(11):956-61. doi: 10.1016/j.biopsych.2012.01.014.

** Ghanizadeh A & Derakhshan N. N-acetylcysteine for treatment of autism, a case report. J Res Med Sci. 2012 Oct;17(10):985-7.

*** Ghanizadeh A. et al. N-acetylcysteine Versus Placebo for Treating Nail Biting, A Double Blind Randomized Placebo Controlled Clinical Trial. Antiinflamm Antiallergy Agents Med Chem. 2013 May 6. [Epub ahead of print]

**** Beloosesky R. et al. Prophylactic maternal N-acetylcysteine in rats prevents maternal inflammation-induced offspring cerebral injury shown on magnetic resonance imaging. Am J Obstet Gynecol. 2013 Mar;208(3):213.e1-6. doi: 10.1016/j.ajog.2013.01.023.

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Ghanizadeh A, & Derakhshan N (2012). N-acetylcysteine for treatment of autism, a case report. Journal of research in medical sciences : the official journal of Isfahan University of Medical Sciences, 17 (10), 985-7 PMID: 23826003

Pristine cysteine-matically done

First of all, sorry for the terrible pun that makes up the post title. What can I say apart from (a) what else rhymes with the amino acid cysteine? and (b) I'd probably make a terrible tabloid newspaper headline maker unlikely to come up with something like this British classic.

In this post I want to focus on a paper by Mostafa Waly and colleagues* (full-text) which includes a couple of notable names on the authorship list including Dick Deth (macroepigenetics and high-fructose corn syrup) and Mady Hornig (carbohydrate digestion and the bacteria which just rolls of the tongue, Sutterella in relation to autism).

The name of the paper's game is cysteine uptake in autism, and how issues with this process may have some interesting connections to "inadequate antioxidant capacity" and onwards affecting prenatal- and postnatal epigenetic programming. I have to admit that this paper does jump around quite a bit in terms of what might impact on what so don't be surprised if I start bringing quite disparate areas into this post. I'll also say now that ultimately this is a paper of mouse models and how an old friend, autoimmuunity, might play some role in cysteine uptake. I'll stress the 'might play some role' before I progress any further.

A few descriptions first:

The amino acid cysteine has cropped up previously on this blog. Not only linked to those very important observations on sulphate (sulfate) levels in various biofluids in cases of autism but also with regards to the growing interest in glutathione (GSH) and autism as a result of cysteine being one of the building blocks of GSH and the various brain revelations published not so long ago.

Epigenetics... well, you could have a look at this post from a few months back introducing epigenetics in relation to autism. The mantra: your genome might not necessarily be your destiny just about covers the science of epigenetics and potentially how epigenetics might resolve some of the issues in the grudge match that is genes vs. environment. I've posted about this elsewhere quite recently (here).

Anyway back to the Waly paper. It is open-access but here are a few of the highlights:

• Unless I am missing something, it is not immediately clear whether this is a summary paper, an experimental-type paper or some combination of the two. After a few reads, I favour the latter option because aside from introducing the important processes involved in cysteine metabolism and epigenetics, there does appear to be some practical experimentation on various types of cell and tissue derived from animal models; in particular the C57BL/6  and SJL/J mouse models. Unfortunately no room for the BTBR Dangermouse model of autism.
• Indeed the practical experiment side of things seemed to involve a few things including: (a) extracting things like regulatory (CD4+ CD25+) T-cells from the mouse models to ascertain the expression of EAAT3, a mediator of cysteine uptake in various body sites (see here) (b) analysis of the level of GSH in the frontal cortex of said mouse models treated with or without the mercury-based preservative thiomersal (or thimerosal) which has been the focus of quite a lot of discussion over the years, and (c) analysis of the activity of methionine synthase, the enzyme that converts homocysteine to methionine, again in the cortex of thiomersal treated and untreated mice.
• A few of the results, but don't quote me on this: GSH levels in the frontal cortex of the SJL/J mice were lower than the C57BL/6 mice. This might make a little more sense if I point you towards some evidence that the SJL/J mouse has been described as quite a good model of autoimmunity, or at least slightly better than the C57BL/6 model.
• Similarly, levels of methionine synthase activity were described as lower in the SJL/J mice.
• It appears that thiomersal treatment had very little effect on GSH or methionine synthase activity results.
• The EAAT3 results, remembering that EAAT3 transports cysteine, cysteine from dietary sources, into cells partly for GSH synthesis. "Expression of EAAT3 was significantly lower in CD4+ T-cells from SJL/J mice versus C57BL6/J mice, suggesting that autoimmunity is associated with impaired capacity for cysteine uptake."

I'll admit that I have scratched my head a few times when reading this paper. The title suggests epigenetic programming to be a core part of the presented evidence but ultimately the data seems to focus more on the speculation around the mouse model differences over any experimental data on a specific epigenetic role tied to autism.

Don't get me wrong, the GSH and methionine synthase expression findings are important and I would love to see how they might compare against the BTBR mouse model of autism bearing in mind its representativeness to autism (see this paper by Pobbe and colleagues** full-text). The additional fact that thiomersal treatment seemed to have very little effect on these parameters in both mouse models is also a potentially important finding.

That being said I almost got the impression that this paper might have been better split into two manuscripts: one on the speculated mechanisms, which provide an excellent overview it has to be said, and another on the fact that C57BL/6 mice don't tend to show as many issues with cysteine, glutathione and methionine pathways as the SJL/J mouse. I caution though that this last finding might not necessarily translate into real life autism.

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* Waly M. et al. Prenatal and postnatal epigenetic programming: implications for GI, immune, and neuronal function in autism. Autism Research & Treatment. 2012
DOI: 10.1155/2012/190930

** Pobbe RL. et al. Expression of social behaviors of C57BL/6J versus BTBR inbred mouse strains in the visible burrow system. Behavioral Brain Research. 2010; 214: 443-449.

Glutathione and autism reloaded

I hope that I don't seem to be treading on old ground with this post on glutathione (GSH) and autism. I have covered some research on a possible link between glutathione and autism spectrum conditions previously (see here) following some interesting suggestions on the potential value of measuring GSH levels as a kind of biomarker for autism. The caveat being that despite an initial AUC=1 much more investigation is required.

This post is slightly different in that I wish to bring to your attention a review paper by Main and colleagues* (full-text) which highlights pretty much what we know, or think we know, about GSH and autism so far. Before progressing I think I should reiterate my caveat of not providing medical or other advice and strongly recommending that a medical physician be involved in all decision making aspects impacting health.

Aside from the content of the paper which I will run through shortly, a familiar name on the authorship list pulled me into this post. Dr Manya Angley, a Pharmacist from the University of South Australia, who has been involved in some really interesting work in autism research (here and here) was part of the team. Avid readers of this blog (if there are any!) might remember my first ever blog post on a paper with some pretty nifty techniques and results based on gut bacterial metabolites turning up in the urine of a group of children with autism. Well, Dr Angley was part of that authorship team also with some impressive company.

The current review carries a few interesting details:

• It pretty much highlights all the work that has been done on measuring glutathione levels in autism up to November 2011. As anyone who has done a systematic review will tell you, this is no mean feat and perfect for answering an undergraduate exam paper such as 'Glutathione and autism: describe and critically discuss'.
• Lower levels of GSH and alterations to chemical relations in cases of autism are a pretty consistent feature of the research carried out so far.
• Serum cysteine levels also seem to be reduced and potentially associated with the severity of presented autistic symptoms. One has to wonder how this might fit into the sulphation issues previously highlighted in cases of autism and whether indeed as in some cases of schizophrenia also, cysteine in the form of NAC might be an area requiring much more serious investigation.
• Serum homocysteine levels in cases of autism don't seem to show significant differences with control values. I was slightly shocked by this finding given previous research. Indeed a more recent paper not included in the current review suggested that hyperhomocysteinemia might be a useful biomarker for autism itself.
• A significant increase in plasma vitamin B6 - pyridoxal-5′-phosphate - was noted in several studies potentially relating to issues with the bioavailability of the vitamin. Perhaps one reason why B6 never lived up to its potential? 

There is quite a lot of other information included in this review which should keep any interested parties content for a few hours. I would hasten to add that in this post I am not uniformly suggesting that everyone with autism has issues with glutathione, cysteine or homocysteine as per my mantra of heterogeneity and comorbidity.

One would however hope that with the continuing development of the third and final NICE guidance for autism in children and young people here in the UK, reviews like this one will figure strongly in informing the panel about potential areas of importance. If anything else this paper should invigorate some real interest into how amino acid and antioxidant chemistry in autism is not just alternative "biomedical" mumbo-jumbo.

To finish, the Carpenters and 'Top of the world'. Have a great day!

* Main PAE. et al. The potential role of the antioxidant and detoxification properties of glutathione in autism spectrum disorders: a systematic review and meta-analysis. Nutrition & Metabolism. April 2012.
DOI: 10.1186/1743-7075-9-35