The gastrointestinal (GI) effects of a gluten- and casein-free diet in autism (continued)

It took a few attempts for me to get this blog post discussing the the paper by Carlo Alessandria and colleagues [1] right. The reasons? Well, predominantly it was because I'm no expert when it comes to the gastrointestinal (GI) tract and autism and, in particular, some of the intricacies of the clinical findings in that context. Don't get me wrong, I am a very keen observer of the peer-reviewed science literature on the bowel and autism (see here and see here  and see here for examples) but I'm no gastroenterologist.

What I did take away from the Alessandria findings is that science is continually looking at the possibility of a link between the various GI issues identified in cases of autism and the still-important peer-reviewed literature on how use of a gluten- and/or casein-free diet (GCFD) seems to have a positive impact for some people on the autism spectrum (see here). Indeed, that there may be lots more to see when it comes to a gut-diet-behaviour interface in relation to (some) autism...

So, slowly does it. First, the aim of the Alessandria study: "evaluating the distribution of human leukocyte antigen (HLA)-DQ2/DQ8 typing among patients with ASD [autism spectrum disorder] with GI symptoms, together with its correlation with duodenal histology and response to GCFD."

HLA-DQ2/DQ8 'typing' are words more commonly found in relation to the prototypical 'dietary gluten can affect health' condition that is coeliac disease. They describe some of the genetics of coeliac disease (CD), and are key components involved in risk for the condition and perhaps other diagnoses of a similar autoimmune ilk. From the 150 or so participants - "with ASD with GI symptoms referred to our outpatient clinic" - who were screened for HLA-DQ2/DQ8, around half were positive (72/151). But researchers did not just stop there. Alongside they also screened for "CD-specific antibodies" (see here and see here for the flavour of what this includes) and concluded that "134 (89%) were negative." To summarise, around half of participants with autism and bowel symptoms possessed the genetics of coeliac disease. But, only around 10% showed a pattern of antibodies related to CD indicative of an immune response to gluten as well as other issues (see here).

And there was more: "Patients were prescribed a 6-month GCFD, and then clinically reassessed." This is where another 'assessment' also becomes relevant to the Alessandria findings. As part of their clinically indicated procedures, participants also underwent endoscopy. This allowed researchers to both look at the inner workings of some of the GI tract and also potentially take biopsy samples. At baseline, before any diet was put in place, they observed that: "56 (37%) showed duodenal microscopic inflammation." 'Duodenal' refers to the duodenum, a part of the GI tract fairly close to the exit of the stomach. Inflammation means just that. And something interesting seemed to connect such bowel findings and dietary response: "Response to diet was related to the presence of histological duodenal alterations at baseline (odds ratio 11.323, 95% confidence interval 1.386-92.549 for Marsh 2 pattern)." In other words, and accepting that correlation is not the same as causation, issues identified in the duodenum - "duodenal histology" - seem to be a possible predictor of response to a gluten- and casein-free diet in relation autistic people.

There is a need for lots more study in this area. Alessandria and colleagues reported their observations on the basis of patients presenting at their clinic with medical needs. This was not a clinical trial in the respect of being randomised (e.g. receiving a diet or not or some other medication to treat such identified bowel issues) or being blinded (researchers and patients not knowing who got what intervention). Knowing a little bit about the use of a GCFD in the context of autism (see here) I'm also acutely aware that 6 months following such a diet is a long time. Even with the best will in the world, some people will not be able to follow such a restrictive diet day-in, day-out. There are issues.

But the Alessandria results are important and promising. They provide a template for further study and an addition to the wealth of biologically-based information on who, on the autism spectrum, might be a 'best candidate' for dietary intervention which excludes gluten and/or casein. I know some people might start up with the 'it's too invasive' arguments in relation to the use of endoscopic and indeed, colonoscopic inquiry when it comes to autism. My counter-argument is that if physicians were presented with a child or adult who did not have autism yet had the same bowel problems as this and other cohorts, would they not be afforded the best healthcare available to them including such inquiry? And why then should a diagnosis of autism but exclusionary to accessing such healthcare? Oh, and it's worth mentioning that at least one of the authors on the Alessandria paper has talked about how technology might eventually make such invasive techniques that little less invasive [2]. Indeed, they've also talked about what else aside from a gluten- and casein-free diet might be clinically indicated for some people on the autism spectrum [3] too with GI issues in mind...

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[1] Alessandria C. et al. HLA-DQ Genotyping, Duodenal Histology, and Response to Exclusion Diet in Autistic Children With Gastrointestinal Symptoms. J Pediatr Gastroenterol Nutr. 2019 Feb 7.

[2] Balzola F. et al. Panenteric IBD-like disease in a patient with regressive autism shown for the first time by the wireless capsule enteroscopy: another piece in the jigsaw of this gut-brain syndrome? Am J Gastroenterol. 2005 Apr;100(4):979-81.

[3] Campion D. et al. The role of microbiota in autism spectrum disorders. Minerva Gastroenterol Dietol. 2018 Dec;64(4):333-350.

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Exclusion diet plus prebiotics for [some] autism?

"To our knowledge, this is the first study where the effect of exclusion diets and prebiotics has been evaluated in autism, showing potential beneficial effects."

So said the results reported by Roberta Grimaldi and colleagues [1] and the findings of their study attempting to "understand the impact of diet on GM [gut microbiota] composition and metabolism in ASD [autism spectrum disorder] children and to investigate the modulating potential of B-GOS® intervention on these parameters."

The study by Grimaldi et al was research registered (see here) and included the 'gold-standard' research design: "A randomised, double-blind, placebo-controlled" study. Researchers divided 30 children diagnosed with an ASD into two groups (A and B), which were subsequently further divided into two groups depending on whether they followed an exclusion diet - "mainly gluten and casein free" - or an unrestricted diet based on the analysis of 4-day food diaries. Within those subdivided A and B groups, half received a placebo supplement of maltodextrin whilst the other half received a "prebiotic B-GOS® mixture" over a 6-week period. As well as including 'run-in', 'beginning of treatment' and 'end of treatment' periods, researchers also included a follow-up period of 2 weeks at the end of the study where various behavioural and physiological measures complemented their use during the experimental period.

Results: there were quite a few results reported as a function of baseline variables such as whether or not participants were following an exclusion diet or not, and as a function of the intervention(s). Behaviourally (probably most importantly), researchers reported that: "Results showed consistent reduction over time in anti-sociability score in children on the combination of the exclusion diet and B-GOS intervention, with the most apparent difference occurring at follow-up." This was measured using the autism 'rising' instrument called the A-TEC (see here) and also complemented other results based on the use of the autism spectrum quotient (AQ). As far as I can make out, all other behavioural and psychometric measures used - the "empathy and systemising quotient (EQ-SQ)... and the Spence’s Children Anxiety Scale-Parent version (SCAS-P)" - did not show any significant changes over the study duration.

Gastrointestinal (GI) symptoms were also examined during the Grimaldi study as per the use of "daily questionnaires for GI function and symptoms" and the utilisation of that fabulous graphical resource, the Bristol stool chart. Authors reported no significant changes/differences as a consequence of intervention, although: "Significantly lower scores of abdominal pain (P < 0.05) and bowel movement (P < 0.001) were reported in children following exclusion diets" at baseline. Interesting, in light of other independent results (see here).

Grimaldi and colleagues also provide quite a lot of data following their examination of fecal and urine samples taken over the course of their investigation. This is perhaps not unexpected given their previous research interests in this area and the proud reputation earned at one of the affiliated institutions. The results? Lots of them. Perhaps the most important intervention-wise was the finding of a "significant increase of Lachnospiraceae family" following B-GOS® intervention. The authors talk about this in terms of the production of butyrate (as per their previous research) and the (positive) reputation this stuff is starting to garner.

Other details? Well, going back to the baseline assessment of samples as a function of the use of an exclusion diet or not, there are some interesting findings. So: "Before prebiotic B-GOS® intervention, we evaluated the nutritional impact of exclusion diets (GFCF) and our results showed deficiency in vitamin D intake, which was significant in children on unrestricted diets." This kinda ties into other research which observed that the 'horror' that is a gluten-free, casein-free (GFCF) diet in relation to autism might actually not be that horrible in nutritional terms (see here). I'm also minded to bring in other recent research which suggested that a GF diet might be a bit of a 'fixer' when it comes to vitamin D deficiency issues under certain clinical circumstances [2]. I say this minus any sweeping generalisations or universal application to the label of autism.

Overall the Grimaldi findings are interesting and suggest further investigations are required on the use of prebiotics and diet in autism; perhaps complementing the still-growing interest in the gut microbiota and autism (see here for one example). Mindful also that diet can itself be seemingly affect gut bacterial composition too [3]. As they stand however, the current results aren't yet a glowing endorsement of B-GOS® intervention 'for' autism, mindful of the small participant group eventually included for study and the relatively short-term nature of the Grimaldi trial. We'll see where this goes... although next time, I'd also like to see a more prominent statement about any harms or side-effects encountered or not during the study period. I assume 'not' in the current study but...

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[1] Grimaldi R. et al. A prebiotic intervention study in children with autism spectrum disorders (ASDs). Microbiome. 2018 Aug 2;6(1):133.

[2] Zingone F. & Ciacci C. The value and significance of 25(OH) and 1,25(OH) vitamin D serum levels in adult coeliac patients: A review of the literature. Dig Liver Dis. 2018 Aug;50(8):757-760.

[3] Berding K. & Donovan SM. Diet Can Impact Microbiota Composition in Children With Autism Spectrum Disorder. Front Neurosci. 2018 Jul 31;12:515.

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Allergy and autism linked (again)

There was something rather timely about the publication of the result by Guifeng Xu and colleagues [1] observing that: "In a nationally representative sample of US children, a significant and positive association of common allergic conditions, in particular food allergy, with ASD [autism spectrum disorder] was found."

Timely, because earlier the same day I read a rather disparaging piece titled 'oversold diets' *trying* to condense down the findings reported by Gogou & Kolios [2]. This duo looked at the "current literature data about the effect of therapeutic diets on autism spectrum disorder", where certain diets are often put in place because of a perceived allergy or intolerance to certain foodstuffs and effects on behaviour and physiology. Gogou & Kolios concluded that: (a) "No serious adverse events have been reported" ('first, do no harm' and all that) and (b) the currently available peer-reviewed data on things like the use of a ketogenic diet and/or a gluten- and casein-free (GFCF) diet 'for' autism is promising but: "More research is needed to provide sounder scientific evidence." I agree that lots more resources and money need to be put into the scientific examination of such diets in the context of autism (see here and see here) including identifying potential best-responders [3]. I'd also like to see gastrointestinal (GI) issues included in the research agenda, in light of other promising peer-reviewed data too on a potential tie-up with said diets and autism (see here)...

Anyhow, back to the Xu findings, which have seemingly attracted some media attention (see here for example). The source of the data was the "National Health Interview Survey [NHIS] collected between 1997 and 2016", a US initiative designed to 'monitor the health of the United States population through the collection and analysis of data on a broad range of health topics'. Including parent or guardian responses on a questionnaire used as part of the NHIS, and specifically the questions: "during the past 12 months, has your child had (1) any kind of food or digestive allergy; (2) any kind of respiratory allergy; (3) eczema or any kind of skin allergy?", data was crunched for nearly 200,000 children.

Authors reported that as a total group (N=199,520), the weighted prevalence of food allergy being reported was about 4%. For a respiratory allergy, the prevalence came in at about 12% and 9% for a skin allergy.

Then to the autism vs. not-autism comparisons where: "A diagnosis of ASD was reported in 1868 children (weighted prevalence, 0.95%; 95% CI, 0.89%-1.01%)." Parents/guardians of those diagnosed with autism were significantly more likely to report all of those various allergies compared to the not-autism group. The figures were: food allergy (autism: 11.25% vs. not-autism: 4.25%), respiratory allergy (autism: 18.7% vs. not-autism: 12%) and skin allergy (autism: 16.8% vs. not-autism: 9.8%). Further: "After adjustment for age, sex, race/ethnicity, family highest education level, family income level, and geographical region, the OR [odds ratio] of ASD was more than doubled (OR, 2.72; 95% CI, 2.26-3.28; P < .001) among children with food allergy compared with those without food allergy." This last observation basically said that there was more chance of autism being reported among those with a food allergy than those without a food allergy.

Caveats? Well, a few to mention. First of all, you'll have noted the words 'parent or guardian responses on a questionnaire' being used, which need to be taken into consideration. No, I'm not casting any aspersions on reporting accuracy of anything like that (parents can be very sensitive to lots of things), but this was not a study of hospital records and tests or anything like that. Second is the use of the word 'allergy'. The medical definition of allergy (see here) is something like 'a biological response to a normally harmless substance'. Allergy is obviously immune-related and typically involves something called IgE (immunoglobulin E) among other things. Whilst some people might think they have an allergy to this, that or t'other, there is still some confusion about whether 'allergy' is the most suitable word in that context without for example, suitable allergy testing being undertaken. I might be being a bit pedantic, but words count...

That all being said, the Xu findings are not the first time that allergy and autism has been raised as being *associated* (see here and see here for examples). Indeed, allergic illness such as something like asthma, has quite a long association with autism (see here) and hints at a possible 'immune-related' connection to at least 'some' autism. That also one of the most common 'comorbid' conditions over-represented in relation to autism - attention-deficit hyperactivity disorder (ADHD) - seems to have an even stronger connection with allergy (see here) is also important to mention, and further strengthens some immune system involvement where there is diagnostic intersection. It also *might* have intervention implications too (see here) (with no medical advice given or intended)...

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[1] Xu G. et al. Association of Food Allergy and Other Allergic Conditions With Autism Spectrum Disorder in Children. JAMA Network Open. 2018; 1: e180279.

[2] Gogou M. & Kolios G. Are therapeutic diets an emerging additional choice in autism spectrum disorder management? World Journal of Pediatrics. 2018. May 30.

[3] Whiteley P. Nutritional management of (some) autism: a case for gluten- and casein-free diets? Proc Nutr Soc. 2015 Aug;74(3):202-7.

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"Comprehensive Nutritional and Dietary Intervention for Autism Spectrum Disorder"

It's been a while coming but the paper by Jim Adams and colleagues [1] detailing the effects of a "comprehensive nutritional and dietary intervention for autism spectrum disorder" has finally seen the peer-reviewed light of day. I say 'a while coming' because as per the ClinicalTrials.gov entry for this research (see here), it was seemingly scheduled to start back in 2011 and be completed by 2013, I assume, without taking 5 years to write up and be published. But better late than never I suppose.

Anyhow, the nutritional and dietary intervention scheduled adopted by Adams et al was rather a complicated affair as per the study description. So: "Day 0: Vitamin/Mineral supplementation begins. Day 30: Essential Fatty Acid supplementation begins. Day 60: Epsom salt baths begin. Day 90: Carnitine Supplementation begins. Day 180: Digestive Enzyme supplementation begins. Day 210: Healthy, casein-free, gluten-free diet [HGCSF] begins." Quite a few of those individual intervention elements have been fodder for this blog before (see here and see here for examples); also reflecting other research interests from Adams and colleagues (see here). Talk about a gluten- and casein-free diet is also music to my [research] ears (see here), as is the welcome inclusion of sulfate / sulphate back into autism research proceedings (see here).

Results of that nutritional and dietary schedule are reported for a starting pool of 67 children diagnosed with an autism spectrum disorder (ASD), where 28 participants completed the 'treatment' arm and some 27 participants completed a non-treatment arm (where no new intervention(s) were reported for the 12 months of the study). Additional findings for 50 not-autism controls (I don't like the word 'neurotypical' and its rather sweeping connotations) are also reported. The study duration was a year, and the sorts of measures examined over the course of the intervention were quite comprehensive, covering both behaviour and cognition and also physiological parameters.

Results: it's always refreshing to see a study first and foremost reports any adverse effects based on the tenet 'first, do no harm'. Authors note that: "A few adverse effects were reported for some treatments" and go on to list what happened over the course of each element of the intervention. They talk for example, how: "One parent reported that implementation of the diet [healthy, gluten- and casein-free diet] in a strict manner resulted in increased aggression towards peers, inability to problem solve, and increased spinning behavior, probably due to frustration in regards to removal of favorite foods." Thankfully, most of the adverse effects noted over the study period were relatively minor and certainly not life threatening. Once again, first, do no harm.

With levels of compliance regarding the various study elements also reported as being quite high, the authors report that across the various behavioural assessments - including the CARS, SRS, VABS, and ATEC - significant effects in favour of intervention were found. Based on blinded evaluations using something called the Reynolds Intellectual Assessment Scales (RIAS), authors reported "a significant improvement in nonverbal intellectual ability in the treatment group compared to the non-treatment group." By contrast, blinded use of the gold-standard assessment instrument known as ADOS revealed "no significant change on the ADOS scores for either treatment or non-treatment group." Interestingly, when parents were asked to rate the effectiveness of each part of the intervention, results revealed that: "The highest rated treatments were the vitamin/mineral supplement and the essential fatty acids, followed by the Healthy HGCSF diets, followed by the carnitine, digestive enzymes, and Epsom salt baths."

Adams and colleagues also provide further details on "3 exceptional cases of improvement during the study, all of which occurred in the treatment group." For one participant it appears that the introduction of a carnitine supplement was associated with some quite remarkable improvements in relation to strength and energy levels in particular. The authors note that: "low carnitine seems likely to have contributed to her challenges, and carnitine supplementation seems to have helped." There could be some interesting tie-ups there with regards to previous peer-reviewed results too (see here for example). For another participant it seemed that the introduction of a HGCSF diet *correlated* with the resolution of urination problems, where a dairy-free diet removed the need for "intermittent catheterization" and resolution of associated problems. OK, these examples don't so much focus on the core issues associated with autism, but I'm pretty sure that they were factors that would have influenced quality of life.

There is quite a bit more to see in the Adams paper and I would encourage readers to take the time to read it in its entirety. Despite the fact that not every measure showed significant effects from such an intervention regime, I like the idea that authors didn't just focus on one intervention but rather, in a systematic way, looked at a whole suite of interventions focused on nutritional and dietary factors. I believe this is more 'naturalistic' in terms of what parents/caregivers tend to report. Indeed the authors themselves discuss how: "A limitation of this study is that all participants received all treatments, whereas probably only a subset are likely to benefit from any single intervention (for example, only participants with low carnitine are likely to benefit from carnitine supplementation)." Yup.

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[1] Adams JB. et al. Comprehensive Nutritional and Dietary Intervention for Autism Spectrum Disorder—A Randomized, Controlled 12-Month Trial. Nutrients. 2018; 10(3): 369.

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Gluten & casein free diet vs. ketogenic diet for autism: Fight!

OK, use of the word 'fight' in the title of this post looking at the study results published by Omnia El-Rashidy and colleagues [1] is a bit melodramatic but the findings do provide some rather interesting talking points when it comes to the whole 'diet and autism' thing (see here). Not least that this is, I think, the first peer-reviewed research occasion when the use of a gluten and casein-free (GFCF) diet in the context of autism has been pitted under experimental conditions against another dietary intervention of some interest, the ketogenic diet. The GFCF diet by the way, involves the removal of foods containing the proteins gluten, found in bread and other cereal-based products and casein, the primary protein found in milk and other dairy products. The ketogenic diet is not a million miles away from the GFCF diet but focuses more on the use of high fat and low carbohydrate foods (see here). Both diets have at least some experimental evidence to suggest that they may impact on autistic and related symptoms at least for some on the autism spectrum. But the evidence is not exactly strong yet [2] for any universal effect(s)...

The results of the El-Rashidy study: both diets seemed to be associated with improvements on various autism-related measures (including the ATEC) when compared with a "third group" who "received balanced nutrition and served as a control group" for 6 months. But on some measures the ketogenic diet (KD) came out on top: "ketogenic scored better results in cognition and sociability compared to GFCF diet group."

The authors rightly note that more needs to be done in this area before any sweeping generalisations are applied. So: "this study is a single center study with a small number of patients and a great deal of additional wide-scale prospective studies are however needed to confirm these results." Add to that issues such as a lack of double-blinding (a real Achilles heel when it comes to dietary intervention research in the context of autism) and there are several more investigations needed in this area; also potentially including measurement of some biological parameters to further elucidate possible mechanisms of effect (see here for one important suggestion). But don't completely disregard all of the peer-reviewed literature in this area as bunk just yet (see here). And that for some on the autism spectrum, there may truly be a diet-sensitive phenotype to examine [3]...

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[1] El-Rashidy O. et al. Ketogenic diet versus gluten free casein free diet in autistic children: a case-control study. Metab Brain Dis. 2017 Aug 14.

[2] Piwowarczyk A. et al. Gluten- and casein-free diet and autism spectrum disorders in children: a systematic review. Eur J Nutr. 2017 Jun 13.

[3] Whiteley P. Nutritional management of (some) autism: a case for gluten- and casein-free diets? Proc Nutr Soc. 2015 Aug;74(3):202-7.

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Time to screen for vitamin D and calcium levels in autism

"The data highlight a gap in calcium and vitamin D supplement prescribing practices among providers caring for children with ASD [autism spectrum disorder] as well as a gap in the practice of checking 25(OH)D levels."

So said research published by Shylaja Srinivasan and colleagues [1] (open-access) with findings tied into an important issue in autism research and clinical practice: potential inequalities when it comes to the provision of suitable healthcare and accessing appropriate screening measures.

Looking at an area of increasing interest when it comes to autism - the sunshine vitamin/hormone that is vitamin D - Srinivasan et al reported that: "Fifty-two percent of the children on the GFCF [gluten-free, casein-free] diet were taking vitamin D supplementation in comparison to 18% of those in the non-GFCF group." Further: "Twenty-four percent of children in the GFCF group had a documented 25(OH)D level compared to none in the non-GFCF group."

If there are any primary messages attached to my musings on this blog, one of them is the growing emphasis on how receipt of a diagnosis of autism or ASD seems to put many people at some enhanced risk of health inequality. I'm not just talking about the idea that autism rarely appears in some sort of diagnostic vacuum (see here) but also that receipt of a diagnosis should not represent the end of screening and/or assessment processes but rather the beginning, and that screening should focus on quite a few things outside of just behaviour. Autisms people, autisms.

Accepting that not everyone might be as enthusiastic as I am about a possible link between vitamin D and autism, Srinivasan and colleagues make some important points about why screening for vitamin D and calcium might be particularly important when it comes to autism. So: "Our results also indicate that only a small proportion of children on the GFCF diet and none on the non-GFCF diet had vitamin D levels checked, even when on multiple medications that could potentially affect vitamin D metabolism and bone health." The types of medication that they are talking about include "antiepileptics, antidepressants, and antipsychotic medications" that "also have influences on bone indices and vitamin D metabolism" some of which have been mentioned previously on this blog (see here).

Insofar as the idea that initiation of a GFCF diet in cases of autism might 'trigger' screening for levels of important nutrients such as calcium and vitamin D in light of removal of dairy products for example, I'm kinda happy to see some data on this. Acknowledging the idea that a GFCF diet for [some] autism (see here) furrows brows in some quarters, I've talked before about how there may be quite a few positives to such intervention insofar as both dietary quality (see here) and where required, the use of supplementary nutrients (see here). This set against a backdrop where issues with food are quite frequently reported alongside autism (see here) and diseases of past years such as scurvy might be making something of a return with the label in mind (see here).

There are still conversations to have about vitamin D and autism and the GFCF diet and autism, of that there is no doubt (see here). The idea however that important health inequalities might follow a diagnosis of autism is something however that cannot be readily ignored. If I've said it once, I've said it a million times: screen. don't assume. screen.

And whilst not trying to make too much of the news that certain groups of people in particular might benefit from vitamin D supplementation, I am inclined to follow the advice of experts when it comes to the extra-skeletal benefits that vitamin D might confer...

To close: it begins... Rogue One.

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[1] Srinivasan S. et al. Calcium and Vitamin D Supplement Prescribing Practices among Providers Caring for Children with Autism Spectrum Disorders: Are We Addressing Bone Health? Autism Research and Treatment. 2016; 6763205.

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Srinivasan, S., O’Rourke, J., Bersche Golas, S., Neumeyer, A., & Misra, M. (2016). Calcium and Vitamin D Supplement Prescribing Practices among Providers Caring for Children with Autism Spectrum Disorders: Are We Addressing Bone Health? Autism Research and Treatment, 2016, 1-6 DOI: 10.1155/2016/6763205

Is a GFCF diet for autism inherently unhealthy? (part 2)

Consider today's entry as a sort of continuation of a previous post looking at the 'horror' that is a gluten-free, casein-free (GFCF) diet for autism (see here).

This time around I'm bringing the paper by Salvador Marí-Bauset and colleagues [1] to your attention and the idea that things might not necessarily be all bad when it comes to the use of a GFCF diet in terms of nutritional quality nor anthropometric values. Indeed, subject to the correct dietetic input, that there may be some important food exchanges going on when a diet devoid of gluten and casein is instigated specifically where an autism diagnosis is mentioned.

I realise that not everyone is as enthusiastic about how food might impact on behaviour and development with at least some autism in mind as I am, but science is coming around to the idea that what we eat (or not) might have some important influences on our being (see here). Appreciating that the GFCF diet is also probably not for everyone [2] (see here also), there continues to be some 'appetite' for such an approach for at least some autism [3]. It is therefore important to understand a little more about what might be the positives and negatives to following such a restrictive dietary regime.

Marí-Bauset et al report results for some 20 children with autism following a GFCF diet compared with 85 "on a regular diet in Valencia (Spain)." This follows a scheme of work from this authorship group looking at various aspects of nutrition when applied to autism [4]. Upon analysing 3-day food diaries, researchers concluded that: "Those on the GFCF diet had a lower weight, body mass index, and total energy, pantothenic acid, calcium, phosphorus and sodium intake." Further however, the GFCF group had: "a higher intake of fiber, legumes, and vegetables" and something of a more favourable fat intake profile that non-GFCF dieters. That last point also ties into other work from the authors [5].

As per the part 1 entry on the nutritional and health related aspects to a GFCF diet for autism (here it is again) there are some details in the Marí-Bauset data that perhaps require some clinical input. I'm thinking specifically about the lower calcium intake in this case, bearing in mind calcium and autism is a very complicated issue (see here) and some continued questioning about the more general link between calcium intake and bone health. The idea that those following a GFCF diet might also present with a lower weight and body mass index (BMI) is also interesting; particularly in light of quite a lot of the chatter in this area focusing on elevated weight and the health effects that can have with autism in mind (see here). I might add that I am in no way endorsing a GFCF diet (or any other diet) for weight loss or management; that's not my job.

The slightly more positive idea that those following a GFCF diet might have a better intake of vegetables and legumes probably also tied into a higher intake of fibre is important. I've previously talked about where the extremes of a limited diet can lead when it comes to [some] autism (see here). Although supplementation has its place in terms of as and when specific deficiencies are present and identified (see here) I think most people would agree that consumption of foodstuffs like fruit and vegetables probably do a better job of supplying nutritional needs than a pill (most of the time). In that respect, one might assume that those on a GFCF diet with more favourable vegetable consumption profile, might be slightly less prone to certain deficiencies. As per other research in this area, we would need a little more biological testing to be sure (see here). The specific idea that fibre intake was higher for the GFCF group is also an important point if one considers fibre to be an essential component when it comes to gastrointestinal (GI) motility, again, as has been specifically mentioned with autism in mind (see here).

In short, and with more research required, the horror that is a GFCF diet for autism might actually with the right clinical input, not be so horrible...

Music: Lily Allen - The Fear.

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[1] Marí-Bauset S. et al. Nutritional Impact of a Gluten-Free Casein-Free Diet in Children with Autism Spectrum Disorder. J Autism Dev Disord. 2015 Oct 1.

[2] Buie T. The relationship of autism and gluten. Clin Ther. 2013 May;35(5):578-83.

[3] Whiteley P. Nutritional management of (some) autism: a case for gluten- and casein-free diets? Proc Nutr Soc. 2015 Aug;74(3):202-7.

[4] Marí-Bauset S. et al. Nutritional status of children with autism spectrum disorders (ASDs): a case-control study. J Autism Dev Disord. 2015 Jan;45(1):203-12.

[5] Marí-Bauset S. et al. Fat intake in children with autism spectrum disorder in the Mediterranean region (Valencia, Spain). Nutr Neurosci. 2015 May 28.

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Marí-Bauset S, Llopis-González A, Zazpe I, Marí-Sanchis A, & Suárez-Varela MM (2015). Nutritional Impact of a Gluten-Free Casein-Free Diet in Children with Autism Spectrum Disorder. Journal of autism and developmental disorders PMID: 26428353

Gluten- and casein-free diets and autism: the Hyman results (at last)

"Although these findings must be interpreted with caution because of the small sample size, the study does not provide evidence to support general use of the GFCF [gluten-free/casein-free] diet."

So said the results of the study finally published by Susan Hyman and colleagues [1] detailing the effects (or not) of a small (n=14) "double-blind, placebo-controlled challenge study" of the use of a diet devoid of gluten and casein for young children diagnosed with an autism spectrum disorder (ASD). If you really want some background history to this often controversial area of autism research, look no further than some of my past musings on this blog (see here and see here) or if you wish, in peer-reviewed form [2].

I say 'finally' in that previous sentence about publication because there has been a considerable degree of waiting for these results to appear in complete peer-reviewed form given that the trial was initially registered in 2004 and things were all supposed to have been wrapped up in 2009 (see here for the ClinicalTrials.gov entry). Some people with their eyes and ears to the autism research grapevine will have probably heard about some of the whys and wherefores of the delay in publishing these results, but I'm not going to get too involved in that here.

Unfortunately the paper is not open-access at the present time but I'll give you a summary of some of the mechanics and findings:

• Some 66 children were initially assessed for eligibility. This was whittled down to 22 kids taking into account those who declined to participate and those who "did not meet inclusion criteria." That inclusion criteria by the way "required children to be enrolled in a comprehensive applied behavior analysis (ABA) intervention program from one of two community agencies" as well as excluding those where seizures were part of clinical presentation and/or the "presence of a chronic illness in addition to ASD that required medical management, celiac disease, documented food allergy to wheat or milk, nutritional compromise such as iron deficiency that required treatment, and family inability to complete rating scales and assessments in English."
• The study design was interesting. It included an implementation phase whereby a GFCF was put in place over the course of 2 weeks (baseline) and maintained for at least 4 weeks. Then came the challenge phase which consisted of weekly challenges to the diet for 12 weeks including one of the following: "foods that contained gluten only, casein only, both gluten and casein, or neither (placebo)." Finally, there was a maintenance period where families were free to "maintain, modify, or abandon the GFCF diet in this phase."
• Various assessments were carried out throughout the study phases covering areas of "physiologic functioning, challenging behaviors (not specific to ASD), and behaviors associated with ASD."
• Results: well, data for 14 of the 22 children were analysed as a function of attrition and or other factors such as "laboratory exclusion criteria." First and foremost we are told: "No serious adverse events were reported during the trial." First, do no harm and all that. When looking at sleep quality and quantity, stool frequency and type, a measure of ADHD (attention-deficit hyperactivity disorder) and a measure of behaviours associated with autism (the Ritvo-Freeman Real Life Rating Scales), authors reported no significant effect following dietary challenges. In other words: "experimental challenges [to the GFCF diet] were not reliably associated with more frequent ASD behaviors." That being said: "All of the families elected to continue the diet for the 12 weeks after completion of the challenges."
• There are some significant strengths to this data based on close monitoring of adherence to the diet, controlling the consumption of gluten and casein levels in challenge snacks and the use of ABA in terms of "stable, consistent educational and behavioral services."
• Likewise however, there are some notable issues associated with the study and the findings outside of the small participant group, not least the emphasis on 'dietary challenge' and importantly the fact that researchers "excluded children who had known gastrointestinal disorders, who might have been more likely to respond positively to dietary restriction." This last point ties in well with other literature in this area [3]. I might add that "individualized supplementation was added for a few participants when deemed necessary by the study dietitian to address low intake of iron, calcium, or vitamin D." Interesting (see here).
• The authors conclude that their study "does not provide evidence to support general use of the GFCF diet" with caveats. 

I can imagine that the Hyman results are probably going to generate some interesting discussions depending on your view of a GFCF diet for autism. I have a professional interest in this topic given some of my research history in this small part of the autism research arena [4] but have tried to stay as objective as possible as per other entries on this topic (see here).

Despite any potential bias I might have, I do still think there is more to see in this area of diet and autism. I've talked before about the idea that the diagnosis of autism is by no means protective against other conditions/labels appearing including those related to issues with gluten for example (see here). This similarly applied to milk also (see here). Some of other peer-reviewed research that I've also been a part of has hinted that there may be 'best responders' to this type of intervention [5] (see here for more discussion); something which ties in well with the concept of plurality and autism (see here).

That caveat about the Hyman study excluding children with known gastrointestinal (GI) disease is also worth re-iterating. Again, this takes autism research into some controversial areas (see here) but as per recent data, both functional (see here) and pathological bowel disorders (see here) do seem to over-represented in cases of autism, so one might see this as an area ripe for further dietary investigations. Indeed, one assumes we might see if there is anything in such an association as and when the Harland Winter trial sees the peer-reviewed light of day (hopefully quite soon).

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[1] Hyman SL. et al. The Gluten-Free/Casein-Free Diet: A Double-Blind Challenge Trial in Children with Autism. Journal of Autism and Developmental Disorders. 2015. Sept 5.

[2] Whiteley P. et al. Gluten- and casein-free dietary intervention for autism spectrum conditions. Front Hum Neurosci. 2013 Jan 4;6:344.

[3] Genuis SJ. & Bouchard TP. Celiac disease presenting as autism. J Child Neurol. 2010 Jan;25(1):114-9.

[4] Whiteley P. et al. The ScanBrit randomised, controlled, single-blind study of a gluten- and casein-free dietary intervention for children with autism spectrum disorders. Nutr Neurosci. 2010 Apr;13(2):87-100.

[5] Pedersen L. et al. Data mining the ScanBrit study of a gluten- and casein-free dietary intervention for children with autism spectrum disorders: behavioural and psychometric measures of dietary response. Nutr Neurosci. 2014 Sep;17(5):207-13.

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Hyman, S., Stewart, P., Foley, J., Cain, U., Peck, R., Morris, D., Wang, H., & Smith, T. (2015). The Gluten-Free/Casein-Free Diet: A Double-Blind Challenge Trial in Children with Autism Journal of Autism and Developmental Disorders DOI: 10.1007/s10803-015-2564-9

Dietary supplementation and autism (and the horror of a GFCF diet)

As perhaps expected, the results reported by Patricia Stewart and colleagues [1] suggesting that: "Few children with ASD [autism spectrum disorder] need most of the micronutrients they are commonly given as supplements, which often leads to excess intake" has generated some interesting media headlines given their mention of the words "gluten/casein-free diet (GFCF)".

A case in point is an article titled: 'Autism Diets' Do Not Provide Children With Adequate Supplementation, Can Lead To Overuse which aside from making little sense (do not provide children with adequate supplementation?) seems to be using the Stewart paper to take pot shots at the mention that GFCF diets have some research (yes, peer-reviewed research) history with autism in mind. Bearing in mind that I have something of an interest in this area, I'm gonna go through some of the findings reported by Stewart et al with a slightly different mindset albeit based on the science to hand.

Drawing on participants with autism (N=288) recruited via the Autism Speaks Autism Treatment Network (ATN) researchers set about examining "dietary supplement use and micronutrient intake in children with ASD." This was accomplished by analysing data from 3-day diet/supplement records "relative to GFCF diet status."

There were a few important results to mention following analysis of [trained] caregiver food/supplement diaries for children based on the reported data and other write-up of the findings:

• Overall, children with ASD were consuming similar amounts of micronutrients as children without ASD. "They also had the same deficits in vitamins D, E, calcium, potassium, and choline as the general pediatric population." I'll come back to some of the details of this shortly.
• Children receiving GFCF diets (~20%) had similar micronutrient intake to those not following a GFCF diet. Indeed: "Children on the GFCF diet consumed more magnesium and vitamin E" and "Children on this diet were more adequately supplemented with vitamin D." These findings hark back to other research suggestions that the 'horror' that is a GFCF diet might not be as poor in nutrition as some people might imagine (see here). I might add that if readers look elsewhere on this blog, you might notice that yours truly has written a book about how a GFCF diet can be implemented safely and nutritiously without the requirement for high levels of supplementation when it comes to autism (sorry for the blatant self-publicity).
• "Dietary supplements, especially multivitamin/minerals, were used by 56% of children with ASD." Using a GFCF diet seemed to be associated with greater supplement use (78% vs 56%) compared with those not following such a diet. I assume this is because of the perception that a GFCF diet is 'nutritionally inadequate' when it comes to important minerals such as calcium for example. That being said: "Calcium supplementation was equally inadequate in those on and off the diet." This is interesting and perhaps relevant to other work on diet, calcium, bone health and autism (see here).
• "Despite different eating behaviors, children with ASD received much of their needed micronutrients from food consumption." The authors suggest that this might have something to do with the various food fortification strategies in place these days, but likewise "may also be responsible for the overconsumption of certain nutrients by children with ASD."
• "Even when supplements are used, careful attention should be given to adequacy of vitamin D and calcium intake." Coming back to the idea that deficiencies noted in this sample of children with autism are not so dissimilar from that seen in the general population, the suggestion that issues such as vitamin D deficiency or insufficiency might be over-represented when it comes to autism continues an emerging research theme (see here).

There are some important points to take from the Stewart paper bearing in mind that 3-day food / supplement diaries are really only providing a snapshot of food / supplement intake and say nothing about the biological levels of various nutrients when it comes to autism. As per the numerous entries on this blog about where eating habits can go wrong with autism in mind (see here) and what happens when vitamin/mineral supplementation in autism is put to the double-blind test (see here) I'd be slightly guarded about drawing too many sweeping conclusions from the new data.

"In clinical practice, each patient needs to be individually assessed for potential nutritional deficiencies or excess." So says Dr Stewart in the press release accompanying the study. I'd be minded to agree with that sentiment given the idea of plurality in autism (see here) and the potential for various biochemistry that can follow a diagnosis (see here and see here) potentially impacting on food and feeding habits. As I've said many times before, receipt of the label of 'autism' or 'autism spectrum disorder' should be a starting point for quite a bit more clinical inspection and not the finishing line...

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[1] Stewart PA. et al. Dietary Supplementation in Children with Autism Spectrum Disorders: Common, Insufficient, and Excessive. Journal of the Academy of Nutrition and Dietetics. 2015. June 4.

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Patricia A. Stewart, Susan L. Hyman, Brianne L. Schmidt, Eric A. Macklin, Ann Reynolds, Cynthia R. Johnson, S. Jill James, & Patricia Manning-Courtney (2015). Dietary Supplementation in Children with Autism Spectrum Disorders: Common, Insufficient, and Excessive Journal of the Academy of Nutrition and Dietetics

Melatonin and leaky gut continued

The paper from Garth Swanson and colleagues [1] reporting findings suggesting that "suppression of melatonin in AD [alcohol use disorder] may promote gut leakiness and endotoxemia" make for the discussion point today.

Continuing a theme from previous times on this blog that "melatonin may serve important gastrointestinal barrier functions" [2] and specifically those related to the concept of intestinal permeability and the so-called 'leaky gut' (see here), these are potentially important findings outside of the more traditional sleep-melatonin link. Swanson et al measured plasma melatonin levels and intestinal permeability via a urine sample in their small participant group of people (as opposed to the previous studies looking at rats). Participants included "20 subjects with alcohol use disorder (AD) and 17 healthy controls (HC, 6 day workers, 11 night workers)."

Authors reported less total sleep time in participants with AD and "and increased fragmentation of sleep." They also suggested that those with AD had lower levels of melatonin and said lower levels "correlated with increased intestinal permeability and a marker of endotoxemia" (i.e. lipopolysaccharide binding protein). Ergo, there may be quite a bit more to see when it comes to melatonin outside of just sleep with the gut in mind.

Wearing my autism research blogging hat and without wishing to equate alcohol use disorder with autism (bearing in mind a diagnosis of autism is in no way protective of such issues developing) I'd like to think that there may be some important science to do in this area on the back of the very preliminary results from Swanson et al. Melatonin as a management agent for sleeping problems in autism [3] is in the ascendancy these days albeit not necessarily working by "simply replacing melatonin" [4]. The various 'molecular handyperson' abilities of melatonin [5] kinda hint that, as one metabolite of the wonderful tryptophan cascade (see here), melatonin might be doing so very much more when taken.

Gastrointestinal or bowel issues being 'over-represented' when it comes to autism is a topic that is becoming less and less argued about these days as a result of the volumes of data being produced on this issue (see here for example). Part of the research question that still needs answering about functional bowel issues and autism is whether or not they are reflective of more 'pathological' states with the gut and in particular, their association (or not) with underlying problems that may well impact on gut barrier function and integrity (see here). The implication being that where present, alterations to gut permeability (leaky gut) may have the ability to do various 'things' to a person including potentially impacting on behaviour and development.

If we assume that melatonin might have some 'gut healing' properties [6] as part of it's repertoire of proposed actions [7] and considering it's growing use with autism in mind, the intriguing prospect that leaky gut is already being 'treated' in cases of autism is opened up. I say this with the understanding that science has yet to fully understand leaky gut or fully corroborate the idea that leaky gut can be treated where present in relation to autism (outside of the data from de Magistris et al [8] suggesting that those with autism "on a reported gluten-casein-free diet had significantly lower IPT [intestinal permeability] values compared with those who were on an unrestricted diet and controls").

So, once again the research gauntlet is thrown down: who will be first to apply some science to this area and measure melatonin vs. placebo in terms of sleep and gut barrier function when it comes to outcome in relation to autism?

Music: The Streets - Dry your eyes.

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[1] Swanson GR. et al. Decreased Melatonin Secretion is Associated with Increased Intestinal Permeability and Marker of Endotoxemia in Alcoholics. Am J Physiol Gastrointest Liver Physiol. 2015 Apr 23: ajpgi.00002.2015.

[2] Sommansson A. et al. Melatonin inhibits alcohol-induced increases in duodenal mucosal permeability in rats in vivo. Am J Physiol Gastrointest Liver Physiol. 2013 Jul 1;305(1):G95-G105.

[3] Veatch OJ. et al. Sleep in Autism Spectrum Disorders. Current Sleep Medicine Reports. 2015. April 24.

[4] Goldman SE. et al. Melatonin in children with autism spectrum disorders: endogenous and pharmacokinetic profiles in relation to sleep. J Autism Dev Disord. 2014 Oct;44(10):2525-35.

[5] Boga JA. et al. Beneficial actions of melatonin in the management of viral infections: a new use for this "molecular handyman"? Rev Med Virol. 2012 Sep;22(5):323-38.

[6] Eliasson L. Melatonin heals the gut. Acta Physiol (Oxf). 2014 Oct;212(2):120-1.

[7] Romo-Nava F. et al. Melatonin attenuates antipsychotic metabolic effects: an eight-week randomized, double-blind, parallel-group, placebo-controlled clinical trial. Bipolar Disord. 2014 Jun;16(4):410-21.

[8] de Magistris L. et al. Alterations of the intestinal barrier in patients with autism spectrum disorders and in their first-degree relatives. J Pediatr Gastroenterol Nutr. 2010 Oct;51(4):418-24.

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Swanson GR, Gorenz A, Shaikh M, Desai V, Forsyth CB, Fogg L, Burgess HJ, & Keshavarzian A (2015). Decreased Melatonin Secretion is Associated with Increased Intestinal Permeability and Marker of Endotoxemia in Alcoholics. American journal of physiology. Gastrointestinal and liver physiology PMID: 25907689

Low bone mineral density and non-coeliac gluten sensitivity and autism?

"An elevated frequency of bone mass loss in NCWS [non-celiac wheat sensitivity] patients was found; this was related to low BMI [body mass index] and was more frequent in patients with NCWS associated with other food sensitivity".

There is no Easter Bunny. There is no Tooth Fairy.
There is no Queen of England.

That was the conclusion reached by Antonio Carroccio and colleagues [1] (open-access) looking at a small group of participants diagnosed with something which seems to fall into a growing spectrum of gluten-related conditions (gluten being the protein found in various cereal crops). The eagle-eyed out there will have spotted how the authors talk about non-celiac 'wheat' sensitivity over and above non-celiac gluten sensitivity as per the idea that: "it is not known what component of wheat causes the symptoms in NCGS patients".

Whilst interesting results, I was actually more intrigued at their possible implications for something like parts of the autism spectrum, on the back of other peer-reviewed research on a possible link with gluten/wheat. I'll take you back to the paper by Ludvigsson and colleagues [2] - discussed in this post - as a starting point and the idea that something not-quite-coeliac-disease (the archetypal autoimmune gluten related condition) might be linked to some cases of autism. The paper by Caio and colleagues [3] (see this post) then suggested that for those presenting with anti-gliadin antibodies as part of NCGS, the use of a gluten-free diet might help dissipate said antibodies, which although not specific to such a scenario with autism in mind, was potentially 'transferable' in light of gluten antibodies being reported in some autism.

Low bone mineral density talked about in the Carroccio paper as being potentially linked to NCWS (or NCGS if you so wish) is also something that has been discussed with autism in mind down the years. I've covered a few papers in this area on this blog, most notably the paper from Hediger and colleagues [4] (see here for the blogpost) and the paper by Neumeyer and colleagues [5] (see here for my take) which indicated that bone mineral density might be lower in cases of autism. Although the gluten (and casein) free diets have been 'blamed' for these results, it appears that nutritional deficiency related to such dietary interventions might not be as serious as some would lead us to expect. If you don't believe me, take a look at my discussion of some peer-reviewed work in this area. The idea also that vitamin D might be lower in quite a few cases of autism is also worthwhile mentioning too bearing in mind the connection between calcium, vitamin and 'strong bones'. Exercise as also playing a role in good bone health also comes into view with autism too.

Marrying the two area together - NCGS and low bone mineral density - with a perspective on at least some autism, may then not seems as outlandish as you might think. Carroccio et al suggest "the role of malnutrition seems very important in our study group" based on their BMI findings. Further they suggest that: "Dietary support should be strongly recommended at the time of NCWS diagnosis, whatever is its pathogenesis." BMI is a bit of mixed bag when it comes to autism as per some of my own work in this area [6] but I'd struggle to argue with their suggestion of appropriate dietary support as and when something like NCGS (or NCWS) is detected on top of a diagnosis of autism.

And then there's gut permeability issues to also consider...

Music: Katie Melua - The Flood.

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[1] Carroccio A. et al. Risk of low bone mineral density and low body mass index in patients with non-celiac wheat-sensitivity: a prospective observation study. BMC Med. 2014 Nov 28;12(1):230.

[2] Ludvigsson JF. et al. A Nationwide Study of the Association Between Celiac Disease and the Risk of Autistic Spectrum Disorders. JAMA Psychiatry. 2013. Sept 25.

[3] Caio G. et al. Effect of gluten free diet on immune response to gliadin in patients with non-celiac gluten sensitivity. BMC Gastroenterol. 2014 Feb 13;14(1):26.

[4] Hediger ML. et al. Reduced bone cortical thickness in boys with autism or autism spectrum disorder. J Autism Dev Disord. 2008 May;38(5):848-56.

[5] Neumeyer AM. et al. Bone density in peripubertal boys with autism spectrum disorders. J Autism Dev Disord. 2013 Jul;43(7):1623-9.

[6] Whiteley P. et al. Body mass index of children from the United Kingdom diagnosed with pervasive developmental disorders. Pediatr Int. 2004 Oct;46(5):531-3.

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Carroccio A, Soresi M, D Alcamo A, Sciumè C, Iacono G, Geraci G, Brusca I, Seidita A, Adragna F, Carta M, & Mansueto P (2014). Risk of low bone mineral density and low body mass index in patients with non-celiac wheat-sensitivity: a prospective observation study. BMC medicine, 12 (1) PMID: 25430806

Metabolomics and autism: the continuing search for biomarkers

I'm always a happy bunny when some of my own research findings receive something like independent replication. So it was when I read the monster paper from Paul West and colleagues [1] (open-access) reporting results based on not one, not two, not three, not even four, but five mass spectrometric methods looking for potential biomarkers for autism. Metabolomics in action (see here for an introduction to this topic).

Rosina @ Wikipedia 

The particular reason for my excitement was the quote: "Creatinine was decreased in children with ASD [autism spectrum disorder] and is consistent with the findings of Whitely et al, who observed similar changes in urinary creatinine in children diagnosed with PDD [pervasive developmental disorder]" based on the results of our paper a few years back [2] (see here for further details of how I spend my spare time talking about urine). The caveat being that we (the Royal We) looked in urine and West et al looked at blood (plasma). I can also forgive the authors for spelling my name wrong too... WHITELEY.

The West paper is open-access but I'm gonna give you a few pointers nonetheless. Stick with me on this one because although quite a long post, this is important work...

• So: "The aim of the study was to perform a broad evaluation of small molecules in blood plasma to discover metabolites that may lead to biomarkers associated with ASD." 
• The value-added bit was that this was study from the MIND Institute which meant that participant groups were very well-defined in terms of diagnosis and presenting symptoms. Indeed, as per other studies of biomarkers (see here), the talk was all about study groups (ASD vs. asymptomatic controls denoted as 'typically developing' TD) and also the use of training and test sets, where: " 82 patient samples (52 ASD and 30 TD samples) were split into two sets, (1) a training set of 61 samples (39 ASD and 22 TD) for identification of statistically significant features and classification modeling and (2) a 21-sample independent validation set (13 ASD and 8 TD) used to evaluate performance of the classification models."
• So, then to the interesting bit... the mass spec methods used and data handling. A combination of liquid chromatography-high resolution mass spectrometry (LC-HRMS) and gas chromatography-mass spectrometry (GC-MS) were used. Actually the LC-HRMS was based on separation using C8 and HILIC column chromatography (the LC part) on both occasions coupled to "electrospray ionization" (the MS part) in positive and negative ion mode so giving "4 separate data acquisitions per sample." That and the GC-MS data makes 5 methods. 
• Various methods/software were used to identify potential metabolites of interest including a couple of programs we use in our lab such as "Agilent Technologies MassHunter Qualitative Analysis software" and the METLIN database.
• Results: as one might imagine, quite a few compounds/metabolites/signals were picked up across the 5 methods used. Table 2 of the paper gives you some idea of the sorts of numbers talked about. That being said, assigning a molecular formula to all those metabolites is rather another matter as per the authors note: "... 179 features comprised 3% of the LC-HRMS and 8% of the GC-MS preprocessed set of features". 'Features' by the way referred to "a moiety detected by the mass spectrometer that is defined by 2 properties 1) the detected mass-to-charge ratio (m/z) and 2) the chromatographic retention time".
• Those 179 'features' formed the basis of the statistical analyses used to try and differentiate autism from control samples. These were subsequently whittled down to: "an 80 feature set [that] exhibited the best combined classification performance metrics... with an average accuracy of 90%, an average sensitivity of 92%, an average specificity of 87%, and an average AUC [area under the curve] of 0.95."
• When moving from training to validation sets, the previous 80 feature model did not work as well. Indeed, some further statistical modelling was used and: "The results suggest that at least 40 features are needed to reach an accuracy of 70% and that a range of 80 to 160 features had the best performance with this independent validation sample set as well as the training set of samples."
• To get to the juicy details of which compounds might be the ones to watch with autism biomarkers in mind, well: "a variety of molecular classes including amino acids, organic acids, sterols, and fatty acids" came up. I've already mentioned creatinine but other prominent mentions were given to "aspartate, glutamate, DHEAS, citric acid, succinic acid, methylhexa-, tetra- and hepta-decanoic acids, isoleucine, glutaric acid, 3-aminoisobutyric acid" and homocitrulline. The authors provide a handy overview of where their results might fit with other autism research areas (e.g. mitochondrial dysfunction, the gut microbiome) which I would encourage interested readers to further peruse. I'm gonna highlight isoleucine as one example where a form of autism has already been talked about with the words 'branched chain amino acids' in mind (see here).
• The authors conclude with a need for quite a bit more study in this area: "This initial study provides proof of concept to further pursue development of metabolic biomarkers of ASD." Personally, I'd like to think that proof-of-concept is perhaps too preliminary a way of introducing metabolomics to autism research given previous research forays (see here and see here and see here) and their potentially important findings. Certainly, things need to be scaled up in terms of participant numbers [3] and also delving into those all-important subgroups of 'the autisms'. Challenges however do remain in assigning molecular formulae to all those compounds detected.

What's more to say? Well, as has been mentioned in a previous post (see here) one always needs to be a little careful when talking about biomarkers for autism as if we're talking about a homogeneous diagnosis and the search for compound X supposedly covering all that heterogeneity (and comorbidity). If we've learned anything from the genetic research on autism for example, it is that simple, universal objective markers are probably not going to be present. Given that the metabolome is to quite a large extent determined by the proteome potentially also intersecting with the microbiome, complexity is probably going to be the keyword.

That being said, I do see merit in the continued use of metabolomics as part of all that system biology kerfuffle (see here) when applied to autism research. I'd personally suggest a few tweaks to how this kind of research is carried out on the basis for example, of not necessarily using the diagnostic label of 'autism' or 'autism spectrum disorder' as a primary starting point. I've talked before on this blog about the notion of best responders and non-responders to intervention for example (see here) and how if one chose to use this as an important variable differentiating those on the spectrum, one might just see a few differences across groups. Such research might also help further inform researchers / clinicians / parents / people on the spectrum who might be best suited for certain types of intervention. Interestingly with dietary intervention in mind, the authors reported that: "Ten of the 52 ASD subjects were on a gluten and/or casein-free (GFCF) diet". Mmm...

Then to some music... Go Your Own Way by Fleetwood Mac. Or if you prefer, the Seaweed version...

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[1] West PR. et al. Metabolomics as a Tool for Discovery of Biomarkers of Autism Spectrum Disorder in the Blood Plasma of Children. PLoS One. 2014 Nov 7;9(11):e112445.

[2] Whiteley P. et al. Spot urinary creatinine excretion in pervasive developmental disorders. Pediatr Int. 2006 Jun;48(3):292-7.

[3] Roessner V. Large sample size in child and adolescent psychiatric research: the way of salvation? European Child & Adolescent Psychiatry. 2014. November 6.

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West, P., Amaral, D., Bais, P., Smith, A., Egnash, L., Ross, M., Palmer, J., Fontaine, B., Conard, K., Corbett, B., Cezar, G., Donley, E., & Burrier, R. (2014). Metabolomics as a Tool for Discovery of Biomarkers of Autism Spectrum Disorder in the Blood Plasma of Children PLoS ONE, 9 (11) DOI: 10.1371/journal.pone.0112445