Autism and the measurement of urinary amino acids

Today's post concerns the findings reported by Aiping Liu and colleagues [1] who, following the analysis of urine samples from a group of children diagnosed with an autism spectrum disorder (ASD) and a not-autism control group, concluded that there may be something to see with regards to the urinary excretion of amino acids.

First things first, amino acids are the building blocks of proteins. Long chains of amino acids form different proteins (and peptides) that serve multiple biological functions. But making up proteins is but one of the roles of amino acids, as a variety of other functions are also included in their repertoire; notably also being the raw material for the formation of some neurotransmitters and related compounds (see here for example).

Liu et al approached their analysis of amino acids in relation to autism from the point of view of their measurement being "potential novel metabolic biomarkers for ASD." This follows something of a trend in autism research circles whereby patterns of certain amino acids and their associated chemistry in certain biofluids might have such 'potential' for some types of autism (see here and see here and see here for some other examples) albeit with certain caveats. Researchers utilised some quite well known methods when it came to their analysis - "liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analysis" - and set to work using a tried-and-tested method (see here): "a two-step discovery–validation approach."

Analysing urine samples from nearly 60 children with autism and over 80 not-autism controls ("28 ASD and 41 TD  [typically developing] children for the discovery stage and from an additional cohort of 29 ASD and 41 TD children for the validation stage"), researchers reported detecting and identifying "63 UAA [urinary amino acid] indicators." Twenty-one of these amino acids and/or amino acid metabolites were observed to be "present at significantly different levels in the urine of ASD children compared with TD children" in both participant sets. These compounds were fairly evenly either higher or lower in the kids with autism group (10 higher and 11 lower). I was particularly interested to see that creatinine was observed to be in the significantly higher category associated with the autism group given some other results that were counter to this finding (see here and see here) including some of my own published data [2] from a few years back. Authors also mention how they "identified a panel of 7 UAA indicators that [most effectively] discriminated between the samples from ASD and TD children (lysine, 2-aminoisobutyric acid, 5-hydroxytryptamine, proline, aspartate, arginine/ornithine, and 4-hydroxyproline)."

From those compounds, a few themes emerged with regards to the biochemistry that *might* show some involvement with autism. So: "Abnormalities in the Methionine Cycle in Children With ASD", "Evidence of High Oxidative Stress Levels in Children With ASD" and "Abnormalities in 5HT Metabolism in Children With ASD" are some of the systems potentially implicated by Liu et al. Needless to say that such biological systems are by no means strangers to autism research (see here and see here for examples) albeit not necessarily always in the same direction as the Liu findings.

Caveats? Well yes, a few, such as a reliance solely on single spot urine samples rather than multiple samples from the same person, no other measures of amino acid content in blood for example, and the focus on participants diagnosed with autism excluding things like "attention-deficit hyperactivity disorder, obsessive compulsive disorder" where 'real-life autism' rarely exists in some sort of diagnostic vacuum (see here). But, the findings are interesting and once again highlight how metabolomics is something particularly valuable to autism research (see here) and complementary to genetic studies for example, when trying to decipher the very heterogeneous autisms (plural). Issues with certain amino acids when identified in the context of autism *might* also point to a wider issue (see here) that could also indicate intervention too...

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[1] Liu A. et al. Altered urinary amino acids in children with autism spectrum disorders. Front. Cell. Neurosci. 2019. Jan 10.

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

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Sulforaphane and autism continued: metabolomics wades in...

"We identified 77 urinary metabolites that were correlated with changes in symptoms, and they clustered into pathways of oxidative stress, amino acid/gut microbiome, neurotransmitters, hormones, and sphingomyelin metabolism."

So said the findings reported by Stephen Bent and colleagues [1] continuing a theme in autism research circles examining the use of a compound called sulforaphane - "a supplement with indirect antioxidant effects that are derived from broccoli sprouts and seeds" - in the context of [some] autism (see here). Once again, I'm sure that there may be people out there with brows furrowing when it comes to talk of a 'broccoli chemical' potentially impacting on the presentation of autism. But peer-reviewed science (placebo-controlled) is peer-reviewed science [2] and not just to be 'put to one side' because it doesn't follow the trends or [research] fashions of the day.

This latest work from Bent et al represents a not-so-methodologically strong attempt (i.e. not placebo-controlled) to bring the science of metabolomics into research proceedings to "examine changes in physiological markers that may underlie beneficial treatment effects from sulforaphane by analyzing changes in urinary metabolites." Metabolomics by the way, as well as being music to my research ears, is something that percolates through quite a lot of autism research these days (see here and see here for examples) as small molecules in urine, blood and other biofluids are separated, detected and elucidated all in the name of science.

A small group of children participated in the Bent study; all had a "formal diagnosis of autism", all were reasonably happy to swallow a tablet containing sulforaphane ("weight-based dosing of sulforaphane") and all were able to provide urine samples before the study started and at the conclusion of the 12 week research period. Parents of participants were also willing and able to complete a couple of behavioural schedules: "the Aberrant Behavior Checklist (ABC) and... the Social Responsiveness Scale (SRS)" at "baseline, 4 weeks, and 12 weeks using an online and secure platform" about their children too.

Results: alongside looking at pre- and post-intervention behavioural scores, the authors also "examined the number of participants who had a clinical response." This is a particularly important detail in the context of autism and the continuing discussions about how the spectrum is well and truly heterogeneous (the autisms?) and so one shouldn't expect every single person diagnosed to somehow have the same genetics and/or biochemistry; also affecting response to any particular intervention. With that in mind, authors also reported that (group) scores on one of their primary outcome measures - the SRS - were significant, indicative of some positive change noted to behaviour over the course of the intervention period. I say this bearing in mind that this was an open-trial, where everyone took sulforaphane and everyone knew that they were taking sulforaphane (including the parents who did the scoring). As for those potential 'best-responders' to sulforaphane use, we are told that: "Eight participants had a clinical response compared to seven who were classified as non-responders."

Then to those metabolomic results, and from a total of nearly 700 compounds identified in urine, approaching 80 of them seemed to show some correlation with the behavioural symptom changes noted. They were put into various categories depending on their biochemical form and/or action, and correlations with behavioural scores (and significance) were presented. Some of the best correlations that I could see were with regards to sphingomyelin metabolism. The authors did seem a bit surprised by results in this area, but added: "It is not clear how sulforaphane might alter sphingomyelin metabolism or availability and whether this is related to clinical benefits, but if this association is confirmed, it has important clinical and treatment implications." I daresay that examination of sphingomyelin metabolism in other contexts (see here for example) might be revealing, particularly in the context of other behavioural/psychiatric labels [3] that could (and do) overlap with autism.

There is a further scheme of work to be followed when it comes to sulforaphane and autism on the basis of these and other scientific results [4]. I'd also suggest that the continued incorporation of the science of metabolomics is a good thing, and adds a further tier of investigation when it comes to studying intervention more generally in the context of autism. It's also an important step in (eventually) coming up with a 'test' for who might be a best-responder to the use of sulforaphane and perhaps associated compounds...

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[1] Bent S. et al. Identification of urinary metabolites that correlate with clinical improvements in children with autism treated with sulforaphane from broccoli. Molecular Autism. 2018; 9: 35.

[2] Singh K. et al. Sulforaphane treatment of autism spectrum disorder (ASD). Proc Natl Acad Sci U S A. 2014 Oct 28;111(43):15550-5.

[3] Castillo RI. et al. From Molecules to the Clinic: Linking Schizophrenia and Metabolic Syndrome through Sphingolipids Metabolism. Frontiers in Neuroscience. 2016;10:488.

[4] Sedlak TW. et al. Sulforaphane Augments Glutathione and Influences Brain Metabolites in Human Subjects: A Clinical Pilot Study. Mol Neuropsychiatry. 2018 May;3(4):214-222.

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"Low parental melatonin levels could be one of the contributors to ASD and possibly ID etiology"

The title heading this post - "Low parental melatonin levels could be one of the contributors to ASD and possibly ID etiology" - comes from the findings reported by Wiebe Braam and colleagues [1]. It continues a research theme on the topic of melatonin and autism or autism spectrum disorder (ASD) (and intellectual disability, ID) (see here).

Perhaps most famously known for its links to circadian rhythms, melatonin is quite an important topic in many areas of autism research and practice. The use of melatonin to influence various sleep issues that seem to be quite regularly present in relation to autism (see here) takes the lion's share of the limelight (see here) but this is a compound potentially doing sooo much more. I've for example, talked about melatonin in relation to intestinal permeability issues (see here) on this blog, as this molecular handyperson [2] offers several possible links/effects with other biological systems. Indeed, Braam et al start from a position where melatonin "is important for normal neurodevelopment and is highly effective in protecting DNA from oxidative damage" continuing an interest for members of the authorship group.

The current findings have, I believe, been seen before on the preprint server bioRxiv [3] so we knew this peer-reviewed publication was coming. Including urine samples from 60 mothers of children diagnosed with autism and a somewhat smaller number from control mothers (with no children diagnosed with autism), researchers submitted said urine samples to analysis for a compound called 6-sulfatoxymelatonin, a primary urinary metabolite of melatonin. I understand that the gold-standard analytical method that is mass spectrometry was the chosen way to determine urinary 6-sulfatoxymelatonin, so one has some confidence that researchers were detecting/measuring this metabolite pretty accurately.

Researchers reported that levels of urinary 6-sulfatoxymelatonin were lower in mums of children diagnosed with autism compared to controls. This finding mirrors that reported in young people with autism [4] and, on more than one research occasion [5]. It looks like there may be a familial element to some of the issues with melatonin which quite neatly fits into other work suggesting similar things with other biological systems including that intestinal permeability [6] that I just mentioned.

Insofar as the implications and 'where next?' questions arising from such work, they are potentially numerous. The relationship with sleep and circadian rhythms is an obvious starting point. Further exploration of whether maternal melatonin biochemistry could be a *risk* factor for similarly altered biochemistry in relation to offspring with autism is also indicated. I'd also like to see some data on paternal and sibling levels of compounds such as 6-sulfatoxymelatonin too (and their related compounds) and how that might correlate with issues such as sleep in those populations.

The inclusion of the word 'sulfate' in 6-sulfatoxymelatonin might also lead to another area of possible research inspection with autism in mind. I hark back to work conducted seemingly aeons ago talking about how sulfate/sulphate chemistry might show some connection to some autism (see here). Sulfation is an important part of the reaction forming 6-sulfatoxymelatonin: 6-hydroxylation --> 6-hydroxymelatonin --> 6-sulfatoxymelatonin (I think), which would be reliant on there being enough sulphate to successfully produce such a reaction on a suitably large scale.

And finally, minus scaremongering or any sweeping generalisations, I do wonder whether further research needs to be done in a wider context of low urinary 6-sulfatoxymelatonin levels. I speak for example of research such as that by Devore and colleagues [7] talking about "Higher urinary melatonin levels... suggestively associated with a lower overall risk of breast cancer." This, in the context that such health issues have been reported with autism mentioned (see here); something which also requires a lot more scientific investigation...

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[1] Braam W. et al. Low maternal melatonin level increases autism spectrum disorder risk in children. Research in Developmental Disabilities. 2018. March 1.

[2] 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.

[3] Braam W. et al. Low parental melatonin levels increases autism spectrum disorder risk in children. bioRvix. 2016. April 2.

[4] Tordjman S. et al. Day and nighttime excretion of 6-sulphatoxymelatonin in adolescents and young adults with autistic disorder. Psychoneuroendocrinology. 2012 Dec;37(12):1990-7.

[5] Tordjman S. et al. Nocturnal excretion of 6-sulphatoxymelatonin in children and adolescents with autistic disorder. Biol Psychiatry. 2005 Jan 15;57(2):134-8

[6] 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.

[7] Devore EE. et al. Urinary Melatonin in Relation to Postmenopausal Breast Cancer Risk According to Melatonin 1 Receptor Status. Cancer Epidemiol Biomarkers Prev. 2017 Mar;26(3):413-419.

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Behind the headline: "Autism: Scientists take 'first steps' towards biological test"

Monday 19th February 2018. I opened my computer up early in the morning and lo and behold, headlines about autism appeared, as exemplified by the BBC article titling this post: "Autism: Scientists take 'first steps' towards biological test." A cold shudder ran down my body as memories of previous 'Super-parenting' improves children's autism and similar headlines sprung to mind and with it, the question of whether big claims were being made...

The paper behind the headlines this time around was from Attia Anwar and colleagues [1] (open-access) and I have to say at first sight I was really rather interested in the specific topic under investigation. Namely: "to explore the diagnostic utility of proteotoxic biomarkers in plasma and urine, plasma protein glycation, oxidation, and nitration adducts, and related glycated, oxidized, and nitrated amino acids (free adducts), for the clinical diagnosis of ASD [autism spectrum disorder]." Interested because the words 'amino acids' (an area of interest to this blog) were mentioned and also that their description of using "stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry" plays to the analytical chemistry nerd that I've seemingly become down the years.

So, bearing in mind the Anwar paper is open-access, what were the hows-and-whys of this 'first steps' research? Well, the initial premise was a sensible one as words like '3-nitrotyrosine (3-NT)' are mentioned following other research noting this compound with some autism in mind (see here). It all ties into the process of oxidative stress and neuroinflammation that are becoming more readily accepted to be part-and-parcel of at least some autism (see here and see here). Researchers set out to see if they could detect some important compounds involved in the process of protein homeostasis (amino acids are the building blocks of protein) and whether, by using the process of machine learning on the derived data (see here for another example of this being applied to autism), the possibility of a diagnostic test for autism might be forthcoming from such work. Yes, this was another example of metabolomics being applied to autism research (see here and see here) and seems to continue a research journey from members of this authorship group [2].

Urine and plasma samples were the chosen analytical media, as authors report on the recruitment of 38 children diagnosed with an ASD and 31 not-autism controls. Yet again, the words 'healthy controls' are used to denote not-autism; something that we really shouldn't be seeing in this day and age. I'd also, by the way, say the same things about the term 'neurotypical' too (see here). The autism group did seem to have quite an extensive diagnostic work-up as both ADOS and CARS scores are presented. Spot blood and urine samples were provided by all and metabolomic analysis was begun...

Results: bearing in mind the significant complexity of both urine and plasma samples when it comes to the presence of small molecules and metabolites, the use of that mass spec method made short work of detecting the glycation markers indicated for study. I note also authors also provide some results on various amino acids in both urine and plasma that is, I think, rather important.

The first thing that struck me was the authors use of a compound called creatinine to correct for sample strength and dilution. I'd like to think I know a thing or two about creatinine (urinary) in relation to some autism on the back of a bit of published research a decade or so back [3]. Our conclusion then and also in some other independent work since (see here) is that caution is required when using creatinine as a corrector with autism in mind...

Having said that, a few findings are noteworthy: "we identified changes in plasma protein AGE [advanced glycation endproducts] and oxidation adducts, increased CML [Nε-carboxymethyl-lysine], CMA [Nω-carboxymethylarginine], and DT [dityrosine] and decreased 3DG-H [3-deoxyglucosone] in ASD." I'm not all too familiar with all of those compounds listed but going back to my observation on oxidative stress being something pertinent to some autism [4], I think there are some important connections to be seen. I note also that at least one of the findings - increased DT residue content of plasma proteins - might also provide a role for those trillions of wee beasties that call us home, the gut microbiota. Mention of the gastrointestinal (GI) tract in the context of autism is likely not to sit well with some people, despite multiple evidence of involvement for some in both a functional sense (see here) and also at a more biological level (see here).

Then to the headline maker: "Algorithms to discriminate between ASD and healthy controls gave strong diagnostic performance with features: plasma protein AGEs—CML, CMA—and 3-deoxyglucosone-derived hydroimidazolone, and oxidative damage marker, DT. The sensitivity, specificity, and receiver operating characteristic area-under-the-curve were 92%, 84%, and 0.94, respectively." Those sensitivity and specificity stats aren't bad at all. They are certainly on a par with other 'classification' attempts with autism in mind, such as when cortisol and a suite of cytokines got the same analytical treatment for example (see here). The trouble is that such stats are based on a relatively small sample size and indeed, children aged between 5-12 years old. Given that many children (but not all) are diagnosed quite a bit earlier than 5 years of age, one has to wonder how relevant any biological test might be at such a later age. One is left feeling that perhaps the discrimination analysis part of the Anwar paper should have perhaps been left until replication on an independent cohort with a larger sample size was carried out, and perhaps relying on more than one testing occasion.

I was also a little bit 'put out' that I couldn't find any 'limitation' discussions in the paper by Anwar et al. There was lots of chatter about how this, that and t'other might relate to biological processes pertinent to some autism but in the discussion section there was very little about what could be wrong with the results as they stand (e.g, small sample sizes). In the current age also when autism is more and more being talked about as NOT being a stand-alone diagnosis (see here) and indeed, probably is a more plural diagnosis ("the autisms"), it is always worthwhile mentioning how any obtained results might have to be framed in those contexts. Yes, authors did have exclusion criteria for study entrance: "Subjects with ascertained medical and neurological comorbidity were excluded, through a medical work up including electroencephalography (recorded during awake and sleep), cerebral magnetic resonance imaging, standard clinical and neurological examination, neurometabolic, and genetic investigations (including comparative genomic hybridization array, molecular assay for Fragile X and MECP2)" but autism pure might not be so typical in the real world [5].

Although some people have reacted quite strongly to the Anwar results, I do think this is quite a good study. No, as it stands, I don't think it can say that autism might be diagnosed on the basis of a urine and/or blood sample. Indeed, we've been here before (see here for example). But it does provide some welcome insight into some of the potential biology associated with at least some autism, and once again, champions the use of some really, really advanced metabolomic technology to provide potential systems biology insights into at least some autism...

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[1] Anwar A. et al. Advanced glycation endproducts, dityrosine and arginine transporter dysfunction in autism - a source of biomarkers for clinical diagnosis. Molecular Autism. 2018; 9: 3.

[2] Anwar A. et al. Quantitation of plasma thiamine, related metabolites and plasma protein oxidative damage markers in children with autism spectrum disorder and healthy controls. Free Radic Res. 2016 Nov;50(sup1):S85-S90.

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

[4] Rossignol DA. & Frye RE. Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism. Front Physiol. 2014 Apr 22;5:150.

[5] Gillberg C. & Fernell E. Autism plus versus autism pure. J Autism Dev Disord. 2014 Dec;44(12):3274-6.

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More bad news for pregnancy paracetamol use?

An article in Medical News Today titled: "Is acetaminophen really safe in pregnancy?" caught my attention recently. Highlighting various recent peer-reviewed research suggesting that pregnancy paracetamol (acetaminophen) use seems to carry some *elevated risk* for quite a few potential adverse events for offspring, the *correlation* news just seems to be getting worse for this go-to over-the-counter painkiller and fever-reducer (see here and see here).

The Medical News Today article highlights a few studies but I want to focus on one in particular, by Carl-Gustaf Bornehag and colleagues [1], which suggested that risk of language delay in female offspring *might* show some correlation with early pregnancy paracetamol use.

Based on data derived from the SELMA study (Swedish Environmental Longitudinal, Mother and child, Asthma and allergy [2]), an important initiative that has already produced quite a bit of data (see here), researchers looked at "prenatal APAP [acetyl-para-aminophenol a.k.a paracetamol] exposure in relation to language development in offspring at 30 months of age." Data from over 750 women were garnered; covering both maternal report on paracetamol usage between conception and enrolment on the study (between 8-13 weeks of pregnancy) and "APAP urinary concentration at enrollment." Yes, this study did actually test for paracetamol [metabolite] concentration(s) in urine during the early stages of pregnancy. Such data was analysed in the context of offspring language development around 30 months of age via a nurse's assessment and parental report on "the number of words the child used (<25, 25–50 and >50)."

Results: paracetamol usage during conception/early pregnancy was not uncommon (around 60% of mothers reported this). Nothing particularly shocking there. Also: "APAP was measurable in all urine samples and urinary APAP was correlated with the number of APAP taken during pregnancy (P < 0.01)." Some good news there that maternal reports of paracetamol usage seemed to correlate pretty well with the measured values of paracetamol metabolites in the urine samples analysed.

Then, although language delay - "parental report of use of fewer than 50 words, termed language delay (LD)" - was present in about 1 in 10 children with a characteristic male bias (see here), female offspring born to mothers reporting paracetamol usage more than six times during early pregnancy were approximately six times more likely to show such language delay than those reporting no paracetamol usage. In effect, the female advantage over males when it came to reported language delay disappeared when paracetamol use was put into the clinical picture. Further: "The OR [odds ratio] for LD in girls whose mothers’ urinary APAP was in the highest compared to the lowest quartile was 10.34 (95% CI 1.37–77.86)." Those girls born to mums with the highest urinary concentrations of paracetamol metabolites, compared with those born to those with some of the lowest levels, seemed also to be at greater risk of language delay.

The authors themselves note that independent replication of their results is required before any grand claims are made. Alongside more formal assessment for something like language delay, I'd also like to see how some of the other metabolites measured for [3] in the SELMA initiative (there we quite a few) might also impact on their conclusions too; particularly in the context of endocrine disrupting chemicals being noted in other studies of paracetamol - offspring development (see here). Further investigation is also required on the 'whys' of paracetamol use in the context of pregnancy: does higher use of something like paracetamol perhaps indicate that some underlying issue that is being 'treated' by such use might also/instead be correlated with offspring developmental outcomes? Pain, fever or something else?

As you can see, the Bornehag study is interesting but not without flaws. Still, set within the rising tide of peer-reviewed research talking about potential issues associated with pregnancy use (excessive use?) of paracetamol and offspring outcomes [4], it's not however something that can be easily ignored...

And whilst we're on the topic of paracetamol, I also recently came across the paper by Abdulaziz Saeedan and colleagues [5] talking about testing the hypothesis that "paracetamol (PCM) can precipitate autistic like features when used to counteract vaccine-induced fever using experimental rat pups." I know (a) this takes us into a 'hot potato' area and (b) this is a study on rats, but set against work from other authors discussing a potential role for post-vaccine paracetamol use in the context of 'risk' of autism [6], such findings also perhaps offer a few further ideas for experimental study.

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[1] Bornehag C-G. et al. Prenatal exposure to acetaminophen and children's language development at 30 months. European Psychiatry. 2018. Jan 10.

[2] Bornehag C-G. et al. The SELMA study: a birth cohort study in Sweden following more than 2000 mother-child pairs. Paediatr Perinat Epidemiol. 2012 Sep;26(5):456-67.

[3] Bornehag C-G. The SELMA study, a longitudinal study following 2,000 mother-child pairs from early pregnancy over birth and up in school age. Environ Health Perspectives. 2013: 5824: S-2-35-05.

[4] Bauer AZ. et al. Prenatal paracetamol exposure and child neurodevelopment: A review. Horm Behav. 2018 Jan 13. pii: S0018-506X(17)30454-3.

[5] Saeedan AS. et al. Effect of early natal supplementation of paracetamol on attenuation of exotoxin/endotoxin induced pyrexia and precipitation of autistic like features in albino rats. Inflammopharmacol. 2018. Jan 11.

[6] Schultz ST. et al. Acetaminophen (paracetamol) use, measles-mumps-rubella vaccination, and autistic disorder: the results of a parent survey. Autism. 2008 May;12(3):293-307.

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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.

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[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.

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Decreased urinary creatinine levels associated with autism (again)

One finding in particular stood out from those reported by Lussu and colleagues [1] following some nifty metabolomic investigations: decreased levels of urinary creatinine in their cohort of participants diagnosed as on the autism spectrum (n=21) compared with "controls (n = 21), these being siblings of autistic patients."

Based on the "use of 1 H-NMR metabolomics to analyze the global biochemical signature of ASD [autism spectrum disorder] patients" researchers turned again to a favourite topic of this blog - metabolomics -  and how the detection and identification of small molecules in various biofluids (urine, blood, saliva, etc) might be particularly informative. Metabolomics is, in essence, all about two things: (i) the analytical technology used to analyse a sample and (ii) the statistical technology used to make sense of the chemical analysis. If one assumes that a biofluid like urine literally contains thousands of compounds and small molecules, you get a flavour for the task facing researchers in this area.

I've talked metabolomics a few times on this blog with autism in mind (see here and see here for examples) but have chosen to zoom in on the Lussu findings in relation to an interesting compound called creatinine. Creatinine is a break-down product of creatine phosphate typically found in muscle. It's generally used as a rough-and-ready guide to the dilution of a urine sample given that it is produced at quite constant rate (see here).

In my day job, I continue to have some interest when it comes to creatinine (urinary) in the context of autism as per some research published a decade or so back [2]. My colleagues and I reported: "Controlling for sample pH and body mass index [BMI], a significant decrease in urinary creatinine concentration was found in the PDD [pervasive developmental disorder] group compared to controls." Other [independent] researchers have also reported similar things [3] when it comes to blood levels of creatinine in the context of autism.

Unfortunately I don't yet have a good explanation as to why creatinine seems to be on the low side when it comes to at least some autism bearing in mind issues such as BMI can seemingly affect values. I do find in interesting that muscle mass may have a bearing on urinary creatinine excretion and could perhaps stretch an association with [some] autism on the basis of more generalised issues with muscle function or tone (see here). That low urinary creatinine *may* also point to issues with kidney function is also interesting and invites quite a bit more study on this organ in relation to autism...

Music: Acceptable in the 80's.

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[1] Lussu M. et al. The urinary 1 H-NMR metabolomics profile of an italian autistic children population and their unaffected siblings. Autism Res. 2017 Mar 11.

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

[3] 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.

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Lussu M, Noto A, Masili A, Rinaldi AC, Dessì A, De Angelis M, De Giacomo A, Fanos V, Atzori L, & Francavilla R (2017). The urinary 1 H-NMR metabolomics profile of an italian autistic children population and their unaffected siblings. Autism research : official journal of the International Society for Autism Research PMID: 28296209

Urinary metabolomics in autism turns up tryptophan (again)

"The tryptophan metabolic pathway collectively displays the largest perturbations in ASD [autism spectrum disorder]."

So said the findings reported by Federica Gevi and colleagues [1] (open-access) who provide yet more 'metabolomic' data when it comes to autism to add to the already quite voluminous peer-reviewed matter on this topic (see here for example).

Just in case you aren't analytical chemistry-saavy, metabolomics is basically the study of the various chemical fingerprints that the multitude of cellular processes going on in the body leave behind. It's the technology available these days that makes metabolomics the discipline that it is, as words such as mass spectrometry and nuclear magnetic resonance (spectroscopy) fill the metabolomic airwaves coupled with some rather smart statistics and software to translate all that captured data into something meaningful.

Gevi et al report results based on the analysis of urine samples from a small-ish group of children diagnosed with an ASD ("idiopathic ASD") compared with samples from a similar number of not autism controls. The aim was to focus on "autistic and unrelated typically developing children 2–8 years old, tightly matched by age, sex, Italian ancestry, and city of origin within the country" and look-see whether a particular HPLC-mass spec technique "hydrophilic interaction chromatography (HILIC)-LC-electrospray ionization (ESI)-MS" might provide some important data on autism vs. not autism.

Results: well, it's always nice to get a research mention in such studies as per the line: "Data were normalized by urinary specific gravity, because creatinine excretion may be abnormally reduced in ASD children" with reference to some work published a few years back [2]. Indeed, this is not the first time creatinine has cropped up in autism metabolomic studies (see here) and is perhaps worthy of quite a bit more study itself (see here).

The authors report that urine samples from those with autism vs. those with not-autism are "largely distinguishable" based on some nifty analysis of the compounds examined from those groups. They even provide a 'top 25 discriminating metabolites' summary to illustrate this fact. Before venturing further into this list, I would perhaps advise some caution however. Caution based on the fact that urine contains many hundreds/thousands of small molecules or chemical entities as a function of being a waste product and carrying waste products from a multitude of different biological processes. It's not outside the realms of possibility that with such a huge number of metabolites, any two groups could be separated out, not just those based on the appearance of autism or not...

Anyhow: "The “metabolome overview” obtained through metabolic pathway analysis (MetPA) shows tryptophan metabolism, purine metabolism, vitamin B6 metabolism, and phenylalanine-tyrosine-tryptophan biosynthesis as the four most perturbed metabolic pathways in ASD." The reference to the aromatic amino acid called tryptophan (the stuff that eventually ends up as serotonin and melatonin) used in the title of this post kinda points to where the money might be when it came to these particular results. I've been interested in tryptophan metabolism and autism for quite a while now (see here for example) and how, outside of the whole serotonin/melatonin bit, there is quite a lot more to see besides. Mention of something called the kynurenine pathway by Gevi is interesting; not least because this pathway overlaps with other conditions/labels too (see here). This pathway might also have some important implications when it comes to epilepsy (see here) as a comorbidity to autism too.

It's also interesting (to me at least!) to note that the authors found something related to the indoles in their analyses too. So: "we also detect a significant increase in indole derivatives of bacterial tryptophan including indolyl 3-acetic acid, indoxyl sulfate, and most prominently, indolyl lactate." Indoxyl sulfate, a uremic toxin - something that is not great for the kidneys - crops up yet again [3] and importantly, highlights how bacteria can also 'go to work' on tryptophan in the gut. Indole -3-acetic acid also brings back research memories in relation to an indole compound close to my research heart, indolyl-3-acrylolyglycine (IAG) [4] that has received a bit of a research bruising quite recently [5] (the authors of that study and another one [6] however, really need to rethink their paper titles insofar as them not actually testing whether dietary intervention actually 'affects' levels of IAG or related metabolites but nonetheless implying so).

There are a range of other findings reported by Gevi and colleagues but I don't want to bore you with all the details. Suffice to say that metabolomics continues its research rise with autism in mind, and provides some rather interesting results. Of course there is more to do in this area; not least the focus on subgroups in these days of 'the autisms' and perhaps a little more metabolomic inquiry when it comes to the myriad of intervention options put forward 'for autism'. Who for example, wouldn't like to see metabolomic profiles pre- and post-folinic acid for example alongside the myriad of other interventions detailed in the peer-reviewed literature? Indeed, I might also advocate a little more investigation on whether specific patterns of urinary compounds might also be related to specific behavioural facets of autism. Given the move towards gut bacteria as potentially showing involvement in some of the results obtained by Gevi et al, it would also be interesting to see if 'altering' certain types of gut bacteria (see here for example) might also have some interesting knock-on effects when it comes to the metabolites detected too? There is quite a bit more to do.

Music and more bad lip reading.... sick of blue milk?

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[1] Gevi F. et al. Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism. Molecular Autism. 2016l 7: 47.

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

[3] Diémé B. et al. Metabolomics Study of Urine in Autism Spectrum Disorders Using a Multiplatform Analytical Methodology. J Proteome Res. 2015 Dec 4;14(12):5273-82.

[4] Bull G. et al. Indolyl-3-acryloylglycine (IAG) is a putative diagnostic urinary marker for autism spectrum disorders. Med Sci Monit. 2003 Oct;9(10):CR422-5.

[5] Wilson J. et al. Can urinary indolylacroylglycine (IAG) levels be used to determine whether children with autism will benefit from dietary intervention? Pediatr Res. 2016 Nov 23.

[6] Dalton NR. et al. Measurement of urine indolylacroylglycine is not useful in the diagnosis or dietary management of autism. Autism Res. 2016 Aug 29.

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Gevi, F., Zolla, L., Gabriele, S., & Persico, A. (2016). Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism Molecular Autism, 7 (1) DOI: 10.1186/s13229-016-0109-5

No association between [current] mycotoxin exposure and autism

Although research stories stating a link (an association if you will) between condition A and factor X make for interesting reading, not all science is so blessed with such news-worthy findings. That's not to say that 'negative findings' are any less important than the 'hey, we found this...' studies, just that they don't perhaps tend to grab the headlines as much as those finding something.

In saying all that I'm standing up for negative findings today and some rather interesting science reported by Jennifer Duringer and colleagues [1] (open-access available here) on the lack of any significant relationship between current urinary mycotoxin content and their cohort of 25 young people diagnosed with an autism spectrum disorder (ASD).

Just in case you didn't already know, mycotoxin refers to "“natural” environmental contaminants, defined as secondary metabolites produced by fungi that reside in our food supply and on every surface in our environment." There are various types of mycotoxins (lots!) that occur as a consequence of various exposure patterns including fungi/moulds growing on foods or being present in our environment. Generally speaking, mycotoxins are something to be avoided (being careful with any sweeping generalisations there).

Duringer et al started their research journey on the basis that weather conditions pertinent to mycotoxin production have been linked to autism (see here) and some small-scale study that "suggested that individual exposure to mold increased the severity of neurophysiological abnormalities seen in autistic children" [2]. I know that furrowed brows will ensue when one talks about precipitation correlating with autism as per 'other correlations' but that was the basis for their study and should so be respected.

The technology used to test participants' urine sample for any presence of mycotoxin is not to be sniffed at as yet again (see here) tandem mass spectrometry steps up to the analytical plate. Some 87 mycotoxins were assayed for based on some previously published work and some important details such as the fact that urine samples were run in triplicate (even six times if you count the fact that each replicate was run in positive and negative ion modes) are included as part of the study protocol.

Results: given the fact that the cohort consisted of 25 young people with autism and 29 age-matched controls, one has to be a little cautious about making too many sweeping generalisations. What we can say is this: 20% of the participants with autism (5/25) came up with a 'positive' sample defined as 'any mycotoxin' being present. This contrasted with 14% of controls (4/29). The amounts detected were low; in some cases very close to or below the limit of quantitation where suitable standards could be obtained and used as comparators. When pooled together, those 9 participants with a positive sample were compared with the participants with a negative result to see if any potential correlates might show statistical significance. They didn't.

There are caveats to this work outside of the small participant group included for study. The authors highlight a particularly important one insofar as their focus on young adults "well after the in utero or infant-toddler exposure window when ASD develops and is diagnosed." In other words, the burden of current exposure to mycotoxins with autism in mind is probably low but that does not mean that "exposure windows" at other times might not exert an important effect. Indeed, other authors have speculated on other correlates [2] linked to the science of epigenetics for example, as perhaps being another area ripe for further study. This should be added to the list.

For now however, we can only base conclusions on the available (peer-reviewed) evidence...

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[1] Duringer J. et al. No Association between Mycotoxin Exposure and Autism: A Pilot Case-Control Study in School-Aged Children. Toxins (Basel). 2016 Jul 20;8(7). pii: E224.

[2] Mezzelani A. et al. Ochratoxin A as possible factor trigging autism and its male prevalence via epigenetic mechanism. Nutr Neurosci. 2016;19(1):43-6.

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Duringer J, Fombonne E, & Craig M (2016). No Association between Mycotoxin Exposure and Autism: A Pilot Case-Control Study in School-Aged Children. Toxins, 8 (7) PMID: 27447670

More [metabolomic] evidence for dysbiosis and some autism?

The paper by Xiyue Xiong and colleagues [1] (open-access available here) took my attention recently and some further evidence contributory to the idea that the trillions of wee beasties that call our gastrointestinal (GI) tract home - collectively known as the gut microbiome - might have some important links to at least 'some' autism.

Describing the results of "a GC/MS based metabolomic approach"  - GC-MS being gas chromatography-mass spectrometry and metabolomic(s) being the analysis of 'small molecule metabolites found in biological fluids such as blood, saliva and urine' - the authors report results based on analysis of urine specimens for some 62 children diagnosed with an autism spectrum disorder (ASD) compared to 62 'not-autism' controls. Bearing in mind that quite a few of the compounds normally found in urine are linked to the goings-on in the gut microbiome, the authors ensured that "Children included in the study had no antianaerobic drug use history" (i.e. certain types of antibiotics were not used).

Results: "Three compounds identified as 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), 3-hydroxyphenylacetic acid (3HPA), and 3-hydroxyhippuric acid (3HHA) were found in higher concentrations in autistic children than in the controls." I was rather interested in the HPHPA finding in particular given that it has previously appeared on this blog in relation to autism and the gut microbiome (see here) following other peer-reviewed findings [2]. The watchword on that previous post was 'dysbiosis' and how alterations in the relative levels of certain gut bacterial species might have some rather intriguing outcomes [3]. The idea therefore being that the action of certain types of bacteria on the proposed starting material for HPHPA (the aromatic amino acids phenylalanine and tyrosine) might influence metabolism and lead to elevations in this metabolite. At this point I'll also refer you to some other musing on research on another aromatic amino acid (tryptophan) that might also be 'autism-relevant' (see here).

Indeed to further test the idea of a gut microbial link to the elevations noted in HPHPA and related metabolites, Xiong et al provide further details: "Fifty HPHPA-positive autistic children (9/50 patients 3HPA-positive and 17/50 patients 3HHA-positive) were selected for oral vancomycin treatment at standard age-appropriate dosages (50 mg/kg/d, 30 days as one therapeutic course) followed by supplement therapy with Bifidobacterium agent (Bifidobacterium BB-12, 2 pills a day)." Use of vancomycin - a quite powerful antibiotic indicated for the treatment of 'Clostridium difficile–associated Disease' [4] among other things - is not unheard of in autism research and practice circles (see here) and this time around there were significant decreases in the levels of HPHPA and related metabolites "which indicated that these compounds may also be from gut Clostridium species." Further, when vancomycin was stopped: "3–6 months later, the concentration of HPHPA almost recovered to its initial level in 3 patients and recovered to 0.08–0.45 times their initial values in 12 patients." Authors also noted that some behavioural scores might have been affected by the use of vancomycin that could be construed along the same lines as when Sandler et al reported on the use of vancomycin with 'regressive-onset autism' in mind [5].

The authors also add in some details about how "measurements of the three metabolites are strong predictors of ASDs and support the potential clinical utility for identifying a subgroup of ASDs subjects in whom disordered phenylalanine metabolism may be a salient characteristic." On this point I'm not convinced that on the basis of 60 or so children and with 3 metabolites in mind (out of the thousands that we excrete everyday influenced by all manner of 'internal' and 'external' forces) there is biomarker potential for 'all autism' just yet. I am in agreement that 'disordered phenylalanine metabolism' for a subgroup on the autism spectrum is a possibility based on the use of 'phenylalanine mopping up' compounds in other peer-reviewed work (see here) for example. But much more research is indicated...

These are interesting results that, yet again, require independent replication. Because I am a bit of stickler when it comes to all-things metabolomics (especially where mass spectrometry is involved) I might be inclined to mention about how adjustment using creatinine might have issues when it comes to autism (see here) which could affect the final quantification of metabolites. I might also suggest that the GC-MS system used and the urine sample pre-treatment applied before analysis could be 'up-graded' taking into account more accurate detection methods (e.g. q-ToF mass spectrometry with liquid chromatography separation) with a greater focus on features like accurate mass.

But don't let me put you off from the idea that marrying metabolomics and microbiomics could be a good autism research idea. Although on the topic of whether we might be able to 'alter' our microbiomes/metabolome in ways other than the use of potent antibiotics, the jury is still out [6] bearing in mind how diet might affect results...

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[1] Xiong X. et al. Urinary 3-(3-Hydroxyphenyl)-3-hydroxypropionic Acid, 3-Hydroxyphenylacetic Acid, and 3-Hydroxyhippuric Acid Are Elevated in Children with Autism Spectrum Disorders. Biomed Res Int. 2016;2016:9485412.

[2] Shaw W. Increased urinary excretion of a 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), an abnormal phenylalanine metabolite of Clostridia spp. in the gastrointestinal tract, in urine samples from patients with autism and schizophrenia. Nutr Neurosci. 2010 Jun;13(3):135-43.

[3] Rogers GB. et al. From gut dysbiosis to altered brain function and mental illness: mechanisms and pathways. Molecular Psychiatry. 2016. April 19.

[4] Shen EP. & Surawicz CM. Current Treatment Options for Severe Clostridium difficile–associated Disease. Gastroenterology & Hepatology. 2008;4(2):134-139.

[5] Sandler RH. et al. Short-term benefit from oral vancomycin treatment of regressive-onset autism. J Child Neurol. 2000 Jul;15(7):429-35.

[6] Kristensen NB. et al. Alterations in fecal microbiota composition by probiotic supplementation in healthy adults: a systematic review of randomized controlled trials. Genome Medicine. 2016; 8: 52.

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Xiong X, Liu D, Wang Y, Zeng T, & Peng Y (2016). Urinary 3-(3-Hydroxyphenyl)-3-hydroxypropionic Acid, 3-Hydroxyphenylacetic Acid, and 3-Hydroxyhippuric Acid Are Elevated in Children with Autism Spectrum Disorders. BioMed research international, 2016 PMID: 27123458

Organophosphate exposure and ADHD?

"Children with higher urinary DMP [dimethylphosphate] concentrations may have a twofold to threefold increased risk of being diagnosed with ADHD [attention-deficit hyperactivity disorder]."

So said the results presented in the paper by Yu and colleagues [1] who looking at "97 doctor-diagnosed ADHD cases and 110 non-ADHD controls who were 4-15 years of age" examined urine and blood samples for various factors including "biomarkers of OP [organophosphate] pesticide exposure." They concluded that, adjusting for creatinine, urine levels of DMP but not other dialkylphosphate (DAP) metabolites were higher in the ADHD group compared with the non-ADHD group. Further: "Organophosphate pesticide exposure may have deleterious effects on children's neurodevelopment, particularly the development of ADHD." At the same time, Yu et al also reported nothing very much to see when it came to blood lead levels (BLLs) between the groups.

This is not the first time that examination of urinary metabolites of OPs have turned up something of a potential relationship with behavioural outcomes related to ADHD. The paper by Bouchard and colleagues [2] also reported a possible connection supporting a "hypothesis that organophosphate exposure, at levels common among US children, may contribute to ADHD prevalence." There too urine was the analytical medium and dialkylphosphate concentrations the target compounds. This and other research looking at this issue have led to statements [3] to the effect that: "Children's exposures to pesticides should be limited as much as possible." I don't think many people would disagree with that sentiment.

I've talked about OPs quite a bit on this blog (see here and see here) and how various conditions/labels might be 'associated' with this class of compounds either when used as insecticides or as something rather more ominous. I've tried not to be too alarmist about the possibility of a connection with health because OPs do serve an important purpose (as an insecticide) and have probably saved quite a few lives as a result. But it is getting increasingly difficult to ignore the possibility that this and other classes of pesticides either alone or in combination with other factors, seem to be implicated in various conditions/labels and more needs to be done looking at the hows and whys. This can however be done without scaremongering.

The Yu results whilst interesting are not however without some cautions. DAP metabolites as markers for OP exposure still requires further investigations [4], not least from which specific OP they are derived from. That other factors such as exposure to second-hand tobacco smoke might also link into the presentation of specific metabolites such as DMP [5] is another consideration. Continuing the theme that combinatorial exposures might also exert an effect [6] other research illustrates how difficult it might be to pin one specific type of exposure to specific behavioural outcomes. And then also we have the added layer of complexity that is the genetics of xenobiotic metabolism with specific focus on OPs. Relationships are likely to be pretty complicated as a result.

Having said all that does not however mean that results like the ones from Yu et al can be just brushed under the carpet...

Music to close, and having watched Guardians of the Galaxy for the Nth time last evening, all I can say is the film soundtrack is kinda cool...

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[1] Yu CJ. et al. Increased risk of attention-deficit/hyperactivity disorder associated with exposure to organophosphate pesticide in Taiwanese children. Andrology. 2016 Apr 12.

[2] Bouchard MF. et al. Attention-deficit/hyperactivity disorder and urinary metabolites of organophosphate pesticides. Pediatrics. 2010 Jun;125(6):e1270-7.

[3] Roberts JR. et al. Pesticide exposure in children. Pediatrics. 2012 Dec;130(6):e1765-88.

[4] Sudakin DL. & Stone DL. Dialkyl phosphates as biomarkers of organophosphates: the current divide between epidemiology and clinical toxicology. Clin Toxicol (Phila). 2011 Nov;49(9):771-81.

[5] Jain RB. Levels of dialkylphosphate metabolites in urine among general U.S. population. Environ Toxicol Pharmacol. 2016 Feb 26;43:74-82.

[6] Osaka A. et al. Exposure characterization of three major insecticide lines in urine of young children in Japan-neonicotinoids, organophosphates, and pyrethroids. Environ Res. 2016 May;147:89-96.

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Yu CJ, Du JC, Chiou HC, Chung MY, Yang W, Chen YS, Fuh MR, Chien LC, Hwang B, & Chen ML (2016). Increased risk of attention-deficit/hyperactivity disorder associated with exposure to organophosphate pesticide in Taiwanese children. Andrology PMID: 27070915

The continued rise of autism research metabolomics

For anyone that has followed this blog down the years you'll probably have noticed that I'm quite a big fan of the inclusion of the science of metabolomics on to the autism research menu (see here for example).

Looking at the myriad of chemical footprints left behind by an almost incomprehensible number of cellular processes, metabolomics offers some real promise to autism in terms of teasing apart phenotypes and as a valuable partner to other -omics sciences in ascertaining the relevance or not of specific biological pathways. All of this set within the context of the plural autisms and the important role of comorbidity (see here).

It is therefore with metabolomics again in mind that I bring to your attention the paper by Binta Dieme and colleagues [1] who weren't joking when they talk about a "multiplatform analytical methodology" with autism in mind. That multiplatform approach included "1H- and 1 H-13C-NMR-based approaches and LC-HRMS-based approaches (ESI+ and ESI- on a HILIC and C18 chromatography column)." If all that sounds like gibberish, the watchwords are NMR - Nuclear magnetic resonance spectroscopy - and LC-HRMS - Liquid chromatography–high resolution mass spectrometry - two of the gold-standard analytical techniques for detecting and identifying compounds of interest in this realm of biology. Some of the other details such as HILIC columns are all to do with how one goes about separating out the individual components of a complicated biological medium like urine as well as some further details about what the authors did to detect them. I might add that this authorship group have some previous form in this area of the autism research landscape (see here).

Based on the analysis of urine samples initially from 22 children with autism and 24 not-autism controls (a training group), researchers talked about the results they obtained from the various metabolomic approaches employed including processing of results by OPLS-DA (orthogonal partial least squares discriminant analysis). I don't want to bore you with the ins-and-outs of what OPLS-DA means (yeah, as if I know!) but suffice to say its all about how one classifies the multitude of data one generates via such analytical methods. This data and analyses were then used to generate a set of compounds (pattern of compounds) potentially predictive of whether or not it could classify a urine sample from someone with autism from a urine sample from someone without autism. Samples from a separate group of participants - "8 autistic children and 8 controls" - were used to 'test' the predictions generated. The authors report that the OPLS-DA model generated "showed an enhanced performance... compared to each analytical modality model, as well as a better predictive capacity (AUC=0.91, p-value 0.006)." AUC by the way, refers to area under the curve and is a term associated with a ROC (receiver operating characteristic). In this respect, the Dieme paper seemed to do pretty well at classifying samples according to autism or not-autism status bearing in mind the relatively small participant group numbers.

Just in case you're not confused enough, there are a few other details about the Dieme paper and findings that are worthy of comment. So: "Metabolites that are most significantly different between autistic and control children (p<0.05) are indoxyl sulfate, N-〈-Acetyl-L-arginine, methyl guanidine and phenylacetylglutamine." Indoxyl sulfate is a particularly interesting compound for quite a few reasons. Not only is the source material for this compound one of the those oh-so-interesting aromatic amino acids, tryptophan (y'know serotonin, melatonin and all that jazz) but the compound itself is described as a uremic toxin [2]. Without wishing to make connections where none may exist, uremic compounds in relation to autism have been discussed before on this blog as per the Elaine Hsiao findings on bacteria and leaky gut in a mouse model of autism (see here) and some chatter about p-cresol and autism (see here and see here). If there is an overlapping factor potentially uniting these findings, it would have to be a possible role for those trillions of wee beasties that call our gut home - the gut microbiome. I might also briefly mention the arginine finding too in relation to a related tryptophan observation for some autism... BH4 (see here).

As I mentioned at the start of this post I am a fan of this area of research area and its potential for furthering knowledge about autism. Larger datasets and perhaps a focus outside of just zooming in on the label of autism are perhaps elements that are needed to aid investigations in this area, alongside a more general combinatorial -omic initiative with a systems biology slant (see here).

Music and I've played this before but here it is again... Weapon Of Choice by Fatboy Slim (a favourite video of my brood).

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[1] Dieme B. et al. Metabolomics study of urine in autism spectrum disorders using a multiplatform analytical methodology. J Proteome Res. 2015 Nov 5.

[2] Vanholder R. et al. The uremic toxicity of indoxyl sulfate and p-cresyl sulfate: a systematic review. J Am Soc Nephrol. 2014 Sep;25(9):1897-907.

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Dieme B, Mavel S, Blasco H, Tripi G, Bonnet-Brilhault F, Malvy J, Bocca C, Andres CR, Nadal-Desbarats L, & Emond P (2015). Metabolomics study of urine in autism spectrum disorders using a multiplatform analytical methodology. Journal of proteome research PMID: 26538324