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...

----------

[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.

----------

Mercury levels and autism meta-analysed

"Indeed, if someone is looking for yet another systematic review and meta-analysis topic, there you go - you're welcome." Those are my words on a previous blogging occasion earlier this year discussing yet another peer-reviewed article on the topic of heavy metals and autism (see here). The findings reported by Tina Jafari and colleagues [1] have seemingly done just that with their meta-analysis of the collected science literature up to June 2017 looking at assessing the "relationship between ASD [autism spectrum disorder] and mercury levels in hair, urine, blood, red blood cells (RBC), and brain."

OK, I know that mention of mercury in the context of autism can be a bit of hot potato in terms of the different types of mercury and their potential sources (see here). I don't want to get into any specific debates on wheres-and-hows in this post but rather focus on what the peer-reviewed science currently says (see here). Jafari et al describe how from the 40+ articles they included for analysis, several themes emerged: (1) blood and brain levels of mercury seemed to be generally elevated in cases of autism vs. controls, and (2) hair levels were typically lower in autism vs controls. Urinary mercury levels were described as "not significantly different" comparing autistic and non-autistic groups although I'll draw your attention to some 'technical talk' in this area that could potentially affect any results produced (see here).

The authors go on to talk about how "detoxification and excretory mechanisms are impaired in ASD patients which lead to accumulation of mercury in the body" which - minus sweeping generalisations - is a conclusion that I've pretty much settled on when talking about some of the findings in this area down the years. There are likely many mechanisms involved in the removal of heavy metals such as mercury from the body but one group in particular, the intersecting "redox and methylation" pathways [2] stand out in view of other research on glutathione levels and autism for example (see here). Other research has pointed to other biological mechanisms that may be worth research consideration [3].

What can and should be done in this area? Well minus any medical or clinical advice given or intended, there are protocols in place as and when mercury poisoning is diagnosed in the general population. There is no reason to assume that these same protocols shouldn't be followed if and when mercury poisoning is diagnosed alongside autism or ASD save any further health inequalities appearing. Autism science needs to also continue it's interest in this area and perhaps make move towards what can be done for example, to 'prop up' biological mechanisms that aid in the detoxification of things like mercury. This may take the form of some rather peculiar research directions (see here) but nonetheless is an area that could potentially be important. Finally, there is the idea that if there are biological issues associated with the removal of several heavy metals including mercury in cases of autism, greater focus on 'avoidance' might also be important. I say this on the basis of findings such as those by Jia Ryu and colleagues [4] who for example, observed that "blood mercury levels at late pregnancy and early childhood were associated with more autistic behaviors in children at 5 years of age." Yes, correlation is not the same as causation, but can we/should we take the chance that the two are connected particularly knowing how detrimental heavy metals can be to human health?

Music to close: Ain't That A Shame by Fats. RIP.

----------

[1] Jafari T. et al. The association between mercury levels and autism spectrum disorders: A systematic review and meta-analysis. J Trace Elem Med Biol. 2017 Dec;44:289-297.

[2] Hodgson NW. et al. Decreased glutathione and elevated hair mercury levels are associated with nutritional deficiency-based autism in Oman. Exp Biol Med (Maywood). 2014 Jun;239(6):697-706.

[3] Gump BB. et al. Background lead and mercury exposures: Psychological and behavioral problems in children. Environmental Research. 2017; 158: 576-582.

[4] Ryu J. et al. Associations of prenatal and early childhood mercury exposure with autistic behaviors at 5 years of age: The Mothers and Children's Environmental Health (MOCEH) study. Science of The Total Environment. 2017; 605-606: 251-257.

----------

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...

----------

[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.

----------

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

NAC for 'social impairment' in youth with autism... probably not

"The results of this trial indicate that NAC [N-acetylcysteine]  treatment was well tolerated, had the expected effect of boosting GSH [glutathione] production, but had no significant impact on social impairment in youth with ASD [autism spectrum disorder]."

So said the results reported by Logan Wink and colleagues [1] (open-access) who, continuing an autism research theme, looked at whether this important L-cysteine prodrug might have more to give when it comes to at least some facets of some autism. Their trial registration entry can be found here. Reporting results from "a 12-week randomized, double-blind, placebo-controlled trial of oral NAC in youth with ASD" researchers followed some 30 children (aged 4-12 years) diagnosed with autism based on their use of NAC or a placebo. Alongside various biological measures including blood levels of "reduced and oxidized glutathione (GSH and GSSG)" and the big 'H' (homocysteine), the primary outcome was the effect on "the CGI-I scale anchored to study physician assessment of core social impairment considering the individuals’ overall level of cognitive, adaptive, and social functioning." I might add that other secondary behavioural outcomes were also included for study.

Bearing in mind the loss (dropping out) of some participants during the study period including details that "three withdrew due to irritability (NAC), diarrhea and encopresis (placebo), and defiant and self-injurious behavior (placebo), respectively" the authors report data on 25 study completers. Titrated doses of NAC depending on tolerance and body weight of participants did seem to do what they were supposed to in terms of an effect on levels of glutathione: "had the expected effect of boosting GSH production in peripheral blood" at 12 weeks.

But... on every other measure - biological and behavioural - there were no significant differences between the NAC and placebo groups leading the authors to conclude that their results "do not support the use of NAC for treatment of core social impairment of ASD." The research door does however remain open as they also comment that: "the health impact of the resultant increase on GSH remains unclear."

These are interesting results. NAC has seemingly found something of a research place in quite a few areas of psychiatry including 'some' autism (see here) and 'some' schizophrenia (see here). The focus has tended to be more on the 'irritability' side of things (see here) which makes it all the more surprising that one of the participants in the Wink trial receiving NAC actually withdrew 'due to irritability'. That being said, the very controlled nature of the Wink study cannot be readily ignored assuming no influence from the placebo [2].

I would like to see a little more on the whole NAC -- cysteine -- glutathione connection given previous discussions with autism in mind (see here). This includes the idea that blood levels of the various compounds involved might not be the same as 'brain levels' and taking into 'level of functioning' into account (see here). But in the context of the Wink data, one has to perhaps realise that NAC is not likely to be a panacea when it comes to autism...

----------

[1] Wink LK. et al. A randomized placebo-controlled pilot study of N-acetylcysteine in youth with autism spectrum disorder. Molecular Autism. 2016; 7:26.

[2] Masi A. et al. Predictors of placebo response in pharmacological and dietary supplement treatment trials in pediatric autism spectrum disorder: a meta-analysis. Transl Psychiatry. 2015 Sep 22;5:e640.

----------

Wink, L., Adams, R., Wang, Z., Klaunig, J., Plawecki, M., Posey, D., McDougle, C., & Erickson, C. (2016). A randomized placebo-controlled pilot study of N-acetylcysteine in youth with autism spectrum disorder Molecular Autism, 7 (1) DOI: 10.1186/s13229-016-0088-6

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.

----------

[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.

----------

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

Acetylcysteine and autism: another case report

I don't want to spend too long on the findings reported by Danielle Stutzman & Julie Dopheide [1] talking about how: "Treatment with acetylcysteine improved ASD [autism spectrum disorder] symptoms, including irritability and aggression, in a teenage patient" but it is a blog-worthy paper.

Describing the experiences of a "7-year-old Hispanic male with ASD and intellectual disability" who was hospitalised due to some rather 'challenging behaviours', the authors noted how the addition of acetylcysteine (often called N-acetlycysteine or NAC for short) seemed to have some pretty interesting positive effects on this young boy's behaviour. Not least also that the use of NAC "was well tolerated, with no observed or reported adverse effects." The authors go on to speculate that within the context of other reports on the use of NAC either alone or as an adjunct medicine, there may be quite a bit more to see with autism in mind, as well as providing some important information about relevant biological pathways in relation to specific 'types' of autism.

I've talked about NAC and autism before on this blog, both within the context of group studies (see here) and under more individual 'N=1' conditions (see here) including with the word 'adjunct' in mind (see here). Within the context of issues that seem to come under the heading of 'challenging behaviours' (bearing in mind the variety of factors that such a description covers) there does appear to be some promising stories coming out of the use of NAC which might have all the be more importance given the lack of good therapeutic interventions for such behaviours.

I'm not at this point going to speculate too much about exactly how and why NAC seems to 'help' when it comes to some challenging behaviours for some people on the autism spectrum. I will suggest that set within the context of studies on glutathione and some autism (see here) there may be some further research to do. That, and not being afraid to look at NAC in relation to something like schizophrenia (see here), and I dare say that there could be surprises for NAC in relation to some autism in future times...

Music to close, and in amongst some recent discussions about 'Where are all the climate change songs?' a gem from The Pixies about a monkey...

----------

[1] Stutzman D. & Dopheide J. Acetylcysteine for treatment of autism spectrum disorder symptoms. Am J Health Syst Pharm. 2015 Nov 15;72(22):1956-9.

----------

Stutzman D, & Dopheide J (2015). Acetylcysteine for treatment of autism spectrum disorder symptoms. American journal of health-system pharmacy : AJHP : official journal of the American Society of Health-System Pharmacists, 72 (22), 1956-9 PMID: 26541950

Brain glutathione and "ASD in intellectually able adult men"

A quote to start:

"[1H]MRS [proton magnetic resonance spectroscopy] measures of cortical and subcortical GSH [glutathione] are not a biomarker for ASD [autism spectrum disorder] in intellectually able adult men."

So said the study published by Alice Durieux and colleagues [1] (open-access available here) based on the measurement of "GSH concentrations in the basal ganglia (BG) and the dorsomedial prefrontal cortex of 21 normally intelligent adult males with ASD and 29 controls who did not differ in age or IQ."

On the basis of there being quite a body of evidence suggesting that functional levels of GSH might be an issue for some on the autism spectrum (see here), researchers set about looking at whether such findings might also translate into 'brain levels' of GSH. Comparing levels of GSH "in unmedicated adult men with ASD and healthy controls of similar age and IQ using [1H]MRS" the authors are pretty clear that the argument that "neuronal damage, secondary to oxidative stress caused by deficient GSH contributes to ASD pathogenesis" might not be universal to all autism.

That being said, I'm not one for sweeping generalisations on this blog and there are caveats to the findings. The authors add for example: "it remains possible that GSH abnormalities exist in other subgroups of individuals with ASD, for example, women, or those with intellectual disability" on the basis of their particular participant selection. The fact also that circulating concentrations of GSH and related metabolites in other tissues (e.g. blood) were not assayed for in the current study means that there may be still more to see in this area including the idea that age might also be an important variable. The brain is also much more than just the BG and prefrontal cortex too.

Although interesting, I'm not yet ready to remove glutathione as being a potentially important compound when it comes to [some] autism just yet. Other studies of post-mortem brain samples with autism and glutathione in mind have not reached the same conclusions as those of Durieux et al accepting that such precious samples are sometimes by no means research perfect. That glutathione issues as part of a wider involvement for oxidative stress in [some] autism might tie into other systems too (see here) invites quite a bit more investigation in this area; perhaps also extending outside of just GSH...

Music: Scissor Sisters - I Don't Feel Like Dancin'.

----------

[1] Durieux AM. et al. Cortical and subcortical glutathione levels in adults with autism spectrum disorder. Autism Res. 2015 Aug 20.

----------

Durieux AM, Horder J, Mendez MA, Egerton A, Williams SC, Wilson CE, Spain D, Murphy C, Robertson D, Barker GJ, Murphy DG, & McAlonan GM (2015). Cortical and subcortical glutathione levels in adults with autism spectrum disorder. Autism research : official journal of the International Society for Autism Research PMID: 26290215

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]...

----------

[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.

----------

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

Cochrane does chelation for autism

Cochrane, in the title of this post, refers to the Cochrane Collaboration and the sterling work done throughout health care synthesising peer-reviewed evidence pertinent to the goal of evidence-based medicine.

The collaboration has recently turned its eye towards the topic of chelation with autism in mind, a topic that has been discussed previously on this blog (see here). Their conclusions based on the findings reported by Stephen James and colleagues [1] (open-access here), looking at the available evidence up to November 2014 are not entirely unexpected: "no clinical trial evidence was found to suggest that pharmaceutical chelation is an effective intervention for ASD [autism spectrum disorder]." Further: "Given prior reports of serious adverse events, such as hypocalcaemia, renal impairment and reported death, the risks of using chelation for ASD currently outweigh proven benefits." A further summary of the findings can be found here.

Based on a search of the peer-reviewed literature on chelation and autism, only one study fulfilled the appropriate criteria (i.e. being randomised), that of Jim Adams and colleagues [2] (open-access here). Actually the Adams study brought about two papers (the second is here [3]) covering both medical and behavioural results following oral DMSA (dimercaptosuccinic acid) therapy in cases of autism. The Adams study reported: "Overall, DMSA therapy seems to be reasonably safe, effective in removing several toxic metals (especially lead), dramatically effective in normalizing RBC [Red Blood Cell] glutathione, and effective in normalizing platelet counts." Further: "Overall, both one and seven rounds of DMSA therapy seems to be reasonably safe in children with ASD who have high urinary excretion of toxic metals, and possibly helpful in reducing some of the symptoms of autism in those children."

The Cochrane review however did not share the optimism. "One trial, which had methodological issues and a relatively small sample size, is insufficient to provide robust evidence on chelation for ASD." James et al also suggested that the Adams study was judged to have "have high or uncertain risk of bias and methodological problems that limited the interpretation of outcomes presented." Even worse: "Given the deleterious effects of chelation, misinterpretation and misuse of the study of Adams et al to justify the use of chelation for ASD is unethical and potentially places children unnecessarily in harm’s way." Ouch.

"At the present time, the theory that heavy metals may cause autism or might worsen symptoms has not been established." I'd agree with this sentiment from James et al but with the proviso that there is ample peer-reviewed evidence to suggest that for at least some on the autism spectrum, the burden of heavy metals does seem to be increased (see here for example). The idea that something like lead (Pb) - a known toxicant adversely affecting child development - might be elevated in [some] cases of autism is of particular importance.

The associated comment by Adams et al on what happened to glutathione levels following DMSA use is something that I'd like to see more science resources put into. Not for one moment do I endorse the use of DMSA without appropriate medical oversight, but knowing what we know about glutathione and autism (see here) and the 'elephant in the room' sentiments, I'd be minded to suggest that there may be quite a bit more to see here particularly in light of the findings from Monin and colleagues [4] talking about glutathione deficits and "myelin maturation".

Music: The Beach Boys - Wouldn't It Be Nice. Well, yes rather.

----------

[1] James S. et al. Chelation for autism spectrum disorder (ASD). Cochrane Database Syst Rev. 2015;5:CD010766.

[2] Adams JB. et al. Safety and efficacy of oral DMSA therapy for children with autism spectrum disorders: Part A--medical results. BMC Clin Pharmacol. 2009 Oct 23;9:16.

[3] Adams JB. et al. Safety and efficacy of oral DMSA therapy for children with autism spectrum disorders: part B - behavioral results. BMC Clin Pharmacol. 2009 Oct 23;9:17.

[4] Monin A. et al. Glutathione deficit impairs myelin maturation: relevance for white matter integrity in schizophrenia patients. Mol Psychiatry. 2015 Jul;20(7):827-38.

----------

James S, Stevenson SW, Silove N, & Williams K (2015). Chelation for autism spectrum disorder (ASD). The Cochrane database of systematic reviews, 5 PMID: 26106752

Cleanrooms and autism?

The paper by Scott Faber and colleagues [1] (open-access) is the topic of today's post and the idea that a cleanroom sleeping environment might impact on some important issues accompanying at least some autism.

Just in case you hadn't come across the concept of a cleanroom, the concept is that a space is provided where various systems are put in place to control environmental pollutants such as dust, microbes and various chemical emissions. More readily associated with the construction of microchips, satellites or the preparation of various pharmaceutics, I was unable to find any previous studies on the use of cleanrooms 'for autism' so the Faber study seems to represent a bit of a first...

The study is open-access but here are a few details:

• Ten children diagnosed with an autism spectrum disorder (ASD) with "prior evidence of heavy metal and chemical toxicity and immune dysregulation seen through low plasma zinc/serum copper ratios and abnormal T and B cell subsets, respectively" were included for study.
• Then: "each child and a parent spent two consecutive weeks sleeping in the cleanroom 
• between May and October 2010." The cleanroom in question was based at The Children's Institute and consisted of HEPA filters, cleanroom bedding, a UV water purification system and was "furnished with ash furniture sealed with water soluble polyurethane." Cleanroom status was confirmed via particle counting.
• Behaviour, blood and hair were collected and tested pre- and post-cleanroom attendance. Blood and hair were analysed for various compounds including nutrients and externally-derived chemicals. Glutathione levels (total, reduced and oxidised) were also assayed for using everyone's favourite analytical method: mass spectrometry.
• Results: children's results were analysed on the basis of their grouping into a younger age cohort (5 and under) and an older age cohort (6 and older). Bearing in mind the small participant numbers: "The overall pattern of results indicates greater improvements in immune dysregulation and behavior in the younger children, age 5 and under." But: "The older children displayed a worsening in behavioral rating scale performance, which may have been caused by the mobilization of toxins from their tissues."
• Among the various results presented, glutathione status seemed to change for at least some of the children over the course of cleanroom residence which the authors interpreted as possibly indicating: "a reduction of oxidative stress." 
• The idea that some children actually presented with increased levels of certain toxicants following their cleanroom experience is an interesting one. "Out of the ten participants, eight decreased mean serum benzene, six increased mean serum toluene and five increased serum xylene." Further: "One possible explanation for these results is that, throughout the study, VOCs were mobilized from tissues into the serum at varying rates."
• The authors conclude: "The performance of a controlled study of 24 hour per day cleanroom exposure for children with autism appears to be an appropriate next step, given the physiologic changes from the limited exposure to a cleanroom environment noted in this study."

Whilst I did find this to be an interesting study, there are a couple of caveats to mention. This was a straight forward open trial of cleanrooms for autism so 'preliminary' is an important word to use in relation to the results. I note also that whilst the trial was registered with ClinicalTrials.org (see here) it seems to have been done pretty retrospectively ("First received: July 16, 2014").

"The child and parent arrived at TCI and spent approximately ten hours each evening in the cleanroom while wearing silk or cotton clothes." What this means is that during the day, when most people are moving around various environments (including the great outdoors) participants were not contained in that sterile environment. There could have been a myriad of different variables to account for the results during this 'free time' including the idea that parents themselves may have altered their child's routines as a function of being part of the study. I say this not to somehow sabotage the results, merely to say that in a study looking at 'detoxification' in relation to autism, one can't discount other factors coming into play.

Insofar as the idea that 'a controlled study of 24 hour per day cleanroom exposure' might be indicated, I think we might have to take a small step back before this is considered. With the limited exposure I've had to cleanrooms during my career I can safely say that I would not take lightly to spending even one day/night in a cleanroom environment. Not least because of the limited living space that would be available as a result of cost considerations but also how the study is potentially talking about housing young children with autism in such an environment and what that might mean for them and an important disruption to their home/school routine among others. That being said I do like one idea discussed by the authors that: "Younger children may have felt an extra closeness to their parents during the study, leading to positive behavioral change." Yes, it's a shocker: children generally love being next to their parents.

Music from Ian Dury & The Blockheads.

-----------

[1] Faber S. et al. A cleanroom sleeping environment’s impact on markers of oxidative stress, immune dysregulation, and behavior in children with autism spectrum disorders. BMC Complementary and Alternative Medicine 2015, 15:71

----------

Faber, S., Zinn, G., Boggess, A., Fahrenholz, T., Kern, J., & Kingston, H. (2015). A cleanroom sleeping environment’s impact on markers of oxidative stress, immune dysregulation, and behavior in children with autism spectrum disorders BMC Complementary and Alternative Medicine, 15 (1) DOI: 10.1186/s12906-015-0564-0

NAC + risperidone = decreased irritability in autism?

It's been a while since I talked about NAC - N-acetylcysteine - on this blog with either autism or schizophrenia in mind (see here and see here respectively). Today I'm going to remedy that situation by bringing the paper by Nikoo and colleagues [1] to your attention, and their observation: "N-acetylcysteine can be considered as an adjuvant therapy for ADs [autistic disorders] with beneficial therapeutic outcomes." Adjuvant therapy by the way, refers to a sort of add-on therapy.

We have to call him, Havok. That's his name now.

Just in case you don't know, NAC among other things is the treatment of choice when it comes to paracetamol (acetaminophen) overdose through it's very important role in the formation of the glutathione (the big 'G' as I should start to call it). Glutathione already has something of a research interest when it comes to autism (see here); more recently increased following papers such as the one by Rahbar and colleagues [2] taking about some of the genetics of the glutathione system with [some] autism in mind, touched upon in a recent post.

Nikoo et al reported results based on a gold-standard randomised, double-blind trial whereby one group of children/adolescents with autism received the antipsychotic risperidone plus NAC and another group received risperidone plus placebo over the course of 10 weeks. Risperidone, as I just mentioned is an antipsychotic medicine which has some interesting history when it comes to [some] autism (see here). Irritability was the focus of the study, and what happened to scores on the "Aberrant Behavior Checklist-Community (ABC-C) Irritability subscale" at baseline (start), 5 weeks and 10 weeks. 

The results suggested that NAC may well have some value as an add-on treatment when it came to scores of irritability in cases of autism as per the authors findings: "By week 10, the NAC group showed significantly more reduction in irritability (P = 0.02) and hyperactivity/noncompliance (P = 0.01) subscales scores."

This is not the first time that NAC + risperidone has been mentioned in the peer-reviewed autism research literature. The paper by Ghanizadeh & Moghimi-Sarani [3] (open-access) also reported significant positive effects albeit alongside a few adverse events such as: "constipation (16.1%), increased appetite (16.1%), fatigue (12.9%), nervousness (12.9%), and daytime drowsiness (12.9%)." This follows other research out of Iran looking at NAC + risperidone in relation to some of the negative symptoms of schizophrenia [4]. On all these research occasions, the experimental period of observation was relatively short (8-10 weeks).

Like many others, I'm always a tad reserved when it comes to the use of antipsychotics for cases/behaviours of/associated with autism. As per my recent discussions on weight gain and such pharmaceutics (see here), one always needs to be a little cautious about the use of such medicines and the application of good medicines management including continual health monitoring as a priority when used. The guidance from NICE here in Blighty advising that such medicines should not be used to manage the core symptoms of autism (see here) is testament to the research base on their effectiveness and their limited place in any management plan. That being said, such pharmaceutics do have a role for some people on the autism spectrum [5] even only if as a 'last resort' in the short-term.

I end by harking back to the paper by Hardan and colleagues [6] talked about in a previous post, which suggested that NAC on it's own might have something to add when it comes to irritability in relation to some autism.  I don't necessarily endorse NAC as being a cure-all for irritability in relation to autism - irritability as part of the so-called challenging behaviours is a very multi-faceted thing with lots of potential precursors [7] - but one might give some consideration to NAC as an intervention for some on the autism spectrum. The next question needs to be: how precisely does it work, and does it have any link back to 'the big G' findings with autism in mind?

Music then. Marvin Gaye and Heard it Through The Grapevine (and something funky is going down apparently).

----------

[1] Nikoo M. et al. N-Acetylcysteine as an Adjunctive Therapy to Risperidone for Treatment of Irritability in Autism: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial of Efficacy and Safety. Clin Neuropharmacol. 2015 Jan 9.

[2] Rahbar MH. et al. Interaction between GSTT1 and GSTP1 allele variants as a risk modulating-factor for autism spectrum disorders. Research in Autism Spectrum Disorders. 2015; 12: 1-9.

[3] Ghanizadeh A. & Moghimi-Sarani E. A randomized double blind placebo controlled clinical trial of N-Acetylcysteine added to risperidone for treating autistic disorders. BMC Psychiatry. 2013 Jul 25;13:196.

[4] Farokhnia M. et al. N-acetylcysteine as an adjunct to risperidone for treatment of negative symptoms in patients with chronic schizophrenia: a randomized, double-blind, placebo-controlled study. Clin Neuropharmacol. 2013 Nov-Dec;36(6):185-92.

[5] Dinnissen M. et al. Clinical and pharmacokinetic evaluation of risperidone for the management of autism spectrum disorder. Expert Opin Drug Metab Toxicol. 2015 Jan;11(1):111-24.

[6] 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.

[7] Guinchat V. et al. Acute behavioral crises in psychiatric inpatients with autism spectrum disorder (ASD): Recognition of concomitant medical or non-ASD psychiatric conditions predicts enhanced improvement. Research in Developmental Disabilities. 2015; 38: 242–255.

----------

Nikoo M, Radnia H, Farokhnia M, Mohammadi MR, & Akhondzadeh S (2015). N-Acetylcysteine as an Adjunctive Therapy to Risperidone for Treatment of Irritability in Autism: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial of Efficacy and Safety. Clinical neuropharmacology PMID: 25580916

Thioredoxin... a new 'diagnosis indicator' for autism?

My name's Buttercup. You've
met Baron von Shush.

"Our study demonstrated that serum TRX [thioredoxin] levels were associated with ASD [autism spectrum disorder], and elevated levels could be considered as a novel, independent diagnosis indicator of ASD." So was the conclusion reported by Qing-biao Zhang and colleagues [1] looking at serum levels of TRX in 80 children diagnosed with an ASD compared against "100 sex and age matched typically developing children".

I'll freely admit that I was not even aware of thioredoxin (TRX) before reading this study; although a quick trawl through some of the research literature on this protein that "act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange" (thank you Wikipedia) hints that I should have been. Quite a good [peer-reviewed] overview of TRX can be found in the paper by Arnér & Holmgren [2] and in particular, describing their role in the process of reducing oxidative stress similar to another compound of interest to this blog: glutathione.

Zhang and colleagues reported significantly higher median serum levels of TRX in their participants with autism compared to asymptomatic controls. Further, that the severity of autism  - as measured using the CARS - might also be linked to TRX levels, and "the optimal cut-off value of serum TRX levels as an indicator for auxiliary diagnosis of autism was projected to be 10.6ng/ml". I might add that such results should not be translated as serum levels of TRX higher than 10.6 ng/ml = autism exclusively, as per other research looking at elevated levels of TRX in other conditions [3].

This is not the first time however that TRX has been studied with autism in mind. The paper by Yusra A Al-Yafee and colleagues [4] (open-access) looking at "sulfur-dependent detoxification mechanisms" in relation to the autism spectrum noted that alongside aberrant values for glutathione (yes, quite consistently so) in their autistic cohort, elevated levels of TRX and related thioredoxin reductase (TrxR) were also detected compared with controls. They stated: "the recorded raised levels of Trx, TrxR and Prxs [peroxidoxins] of the present study could be related to... previous work... which they proved that Saudi autistic children are under H2O2 stress due to over expression of SOD and a slightly lower activity of catalase." Interestingly, this group also suggested that TRX and glutathione parameters might also have some legs when it comes to their usefulness "as diagnostic biomarkers of autism."

Oxidative stress and autism is a research area in the ascendancy. With links being made to the gastrointestinal (GI) issues quite commonly reported alongside a diagnosis of autism (see here) and some really quite interesting work talking about oxidative stress potentially inducing mitochondrial issues in autism (see here), some important correlations are being made. My recent discussions including the paper by Main and colleagues [5] (open-access) suggesting that: "children with autism are more sensitive to necrosis caused by oxidative and nitrosative stress than their non-autistic siblings" adds to the intrigue, as does a little study about broccoli extracts recently...

Of course, quite a lot more replicative work is required, also including more focus on the hows and whys of issues with redox regulation related to autism. But more and more, oxidative stress is taking a place among quite a few other issues detected in at least some cases of autism [6].

Music to close: Queenie Eye by Paul McCartney.

----------

[1] Zhang QB. et al. Thioredoxin: A novel, independent diagnosis marker in children with autism. Int J Dev Neurosci. 2014 Nov 26. pii: S0736-5748(14)00191-9.

[2] Arnér ES. & Holmgren A. Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem. 2000 Oct;267(20):6102-9.

[3] Yamada Y. et al. Elevated serum levels of thioredoxin in patients with acute exacerbation of asthma. Immunol Lett. 2003 Apr 3;86(2):199-205.

[4] Al-Yafee YA. et al. Novel metabolic biomarkers related to sulfur-dependent detoxification pathways in autistic patients of Saudi Arabia. BMC Neurol. 2011 Nov 4;11:139.

[5] Main PA. et al. Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress. Mutagenesis. 2013 Jul;28(4):475-84.

[6] Rossignol DA, Frye RE. Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism. Frontiers in Physiology 2014;5:150. doi:10.3389/fphys.2014.00150.

----------

Zhang QB, Gao SJ, & Zhao HX (2014). Thioredoxin: A novel, independent diagnosis marker in children with autism. International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience PMID: 25433158

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.

----------

[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.

----------

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