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

Autism and the GFCF diet: ScanBrit episode 2

"These preliminary observations on potential best responder characteristics to a gluten- and casein-free diet for children with autism require independent replication".

That sentence, taken from a recent (pre-print) publication I was very peripherally involved in writing, is probably the most important thing to take from the paper by Lennart Pedersen and colleagues* and certainly is a message that I would be very keen to promote.

Skandinavism @ Wikipedia 

ScanBrit, was/is a meeting of minds between the group I work with and other research groups based in Denmark and Norway, who wanted to experimentally examine the question of whether a diet devoid of foods containing gluten and casein might be able to impact on the presentation of autism in children.

Combined with a small but growing bank of research, the net outcome was that science should perhaps be looking at a possible dietary effect at least in relation to some cases of autism. One of the main issues being that the evidence base (including our own results**) was still limited in terms of the methodological quality of the investigations*** being undertaken (see here for a review). Oh and mechanism-wise, (i.e. how and why did diet 'work'?) we are still feeling around the edges, despite a few avenues of potential further inquiry (see here and here) including revisiting some older ideas.

In the most recent paper, (we) the ScanBrit collaboration were looking to further analyse the wealth of data which was generated over the course of the 2 years of experimental study and in particular, try and ascertain whether there were certain characteristics which might be markers for dietary response or not. As any person following the GFCF diet or their parents know, this is not an easy diet to follow and might, under certain circumstances, place a person at risk of nutritional deficiencies (see here) and/or other more socially-mediated issues (see here). Identifying markers of who might be a responder or non-responder would therefore save quite a lot of time, effort and expense.

Interestingly, as I've indicated in a previous post, some of the most significant and consistent behavioural changes which were noted in our original trial as correlating with the installation of a gluten- and casein-free (GFCF) diet were actually outside of the core symptoms of autism; instead potentially reflecting changes to attention and hyperactivity, more traditionally linked to the symptoms of attention-deficit (hyperactivity) disorder (ADHD). In other words, the effect of a GFCF diet might be more peripheral than anything else in cases of autism, in terms of targeting issues associated with ADHD which might also be present alongside the core autism symptoms (see my ESSENCE post and the very interesting paper by Sprenger and colleagues****).

Chronological age was identified as potentially being a factor influencing response to the GFCF diet, and in particular, those children aged between 7 and 9 years old as being potential best responders. I have to say that I am still mystified by this finding. Traditionally, I'd always assumed that there was a 'younger, the better' sentiment when applied to the possible effectiveness of dietary intervention on cases of autism, as per the rising tide of literature on other early interventions. Outside of the relatively small participant numbers we included for study, this could mean that there is some tie-in with the ADHD symptoms which seem to be targeted, bearing in mind my relative inexperience when talking about the manifestation of ADHD throughout childhood. In light of the recent case study from Dr Martha Herbert on a GFCF diet morphing into a ketogenic diet (see here), I do wonder if there might be other comorbidities also being affected which might also have some tie-in too. Who knows.

Again, without making too much of this variable at the current time, there was also a possibility for some involvement of one of the urinary compounds***** we focused on in the original ScanBrit trial as potentially being involved in response. I know biomarkers and autism is still a very complicated subject (see here) bearing in mind the heterogeneity of presentation and the potential influence of all that heightened risk of comorbidity. That all being said, trans-indolylacryloylglycine (IAG) whilst not being a biomarker for autism****** continues to garner interest with its possible connection to gastrointestinal (GI) comorbidity*******. More investigation needed here methinks.

I don't think we have made any startling discoveries with the publication of this paper. We were limited to the variables that we examined over the course of the study and so were unable to talk about whether other, more diet-relevant factors such as coeliac disease (CD) or related pathology - non-coeliac gluten sensitivity for example - might also be involved in response to diet. This was a major shortcoming of our original paper I'll freely admit.

That we found that inattention and hyperactivity might be key factors in dietary response however, is to my mind, potentially very important. That targeting such behavioural issues might also have a knock-on effect to more core autism presentation (as per our ADOS results on communication) is also an area requiring further investigation and perhaps reflects how autism (or should that be the autisms) is so very much more than just the formal clinical descriptions we give it. RDoC anyone?

Blogging about ones own peer-reviewed research is something that I've always been a little bit hesitant about doing. Aside from the possible charge of self-publicity and the natural tendency to think that yours is obviously an important paper (who wouldn't think that about their own work?), one might argue that important features such as the objectivity required for good science blogging, might to some degree be compromised. Sort of like marking your own homework as per one of my colleagues oft-cited phrases. I very much hope that I've not over-stepped the mark on good science blogging in this post on Lennart's paper. Indeed I'll end by reiterating part of the opening sentence to this post on the requirement for independent replication before anyone gets too excited about these results, as indeed the dietary research base as it presently stands is in need of more inquiry********.

And since we're on the topic of diet and autism, would that be one hump or two*********?

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* 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. 2013 (pre-print)

** 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.

*** Millward C. et al. Gluten- and casein-free diets for autistic spectrum disorder. Cochrane Database Syst Rev. 2008 Apr 16;(2):CD003498.

**** Sprenger L. et al. Impact of ADHD symptoms on autism spectrum disorder symptom severity. Res Dev Disabil. 2013 Aug 21;34(10):3545-3552.

***** Anderson RJ. et al. Identification of indolyl-3-acryloylglycine in the urine of people with autism. J Pharm Pharmacol. 2002 Feb;54(2):295-8.

****** Wright B. et al. Is the presence of urinary indolyl-3-acryloylglycine associated with autism spectrum disorder? Dev Med Child Neurol. 2005 Mar;47(3):190-2.

******* Wang L. et al. Is urinary indolyl-3-acryloylglycine a biomarker for autism with gastrointestinal symptoms? Biomarkers. 2009 Dec;14(8):596-603.

******** Winburn E. et al. Parents' and Child Health Professionals' Attitudes Towards Dietary Interventions for Children with Autism Spectrum Disorders. J Autism Dev Disord. 2013 Sep 1.

********* Al-Ayadhi LY. & Elyass Elamin N. Camel Milk as a Potential Therapy as an Antioxidant in Autism Spectrum Disorder (ASD). Evidence-Based Complementary and Alternative Medicine, 2013, Article ID 602834, 8 pages, 2013. doi:10.1155/2013/602834

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Lennart Pedersen, Sarah Parlar, Kajsa Kvist, Paul Whiteley, & Paul Shattock (2013). 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 Nutritional Neuroscience

Autism, aromatic amino acids and gut bacteria: a hypothesis

Mass-ive @ Paul Whiteley

The publication of an interesting article by T. Andrew Clayton* in FEBS Letters (full-text) with a speculative hypothesis regarding the amino acid phenylalanine, gut bacteria and autism has my full attention. A quick search for the author suggests quite an accomplished research career with a scientific publication track-record to boot particularly in the area of metabolomics and with some very distinguished company. That and mention of one Prof. Glenn Gibson in the acknowledgements leads me to believe that this chap knows what he is talking about.

This paper was always going to attract my attention because it made reference to the compound trans-indolylacryloylglycine or IAG and autism. What on earth is IAG I hear you ask? Well it's a compound, a peculiar metabolite of the amino acid tryptophan, that has surrounded the research I have been involved in for many years both from a detection and possible biological marker point of view. As it happens, the initial speculation on IAG potentially being a more general biomarker for autism has subsided as per this article and this article but that's not to say that it may not be relevant to some groups on the autism spectrum, particularly those who also present with comorbid gastrointestinal issues. Indeed there still remains the possibility that there may be some 'relationship' between IAG, gluten and the so-called leaky gut (more on this later).

The crux of the current paper is that another amino acid, phenylalanine, which shares more than a passing relationship with other aromatic amino acids particularly tryptophan and tyrosine in terms of the 'give-and-take' ability of the respective hydroxylase enzymes and the shared use of cofactors such as BH4, is influenced by certain types of gastrointestinal bacteria. This in turn might also explain some of the origins of IAG, derived from tryptophan.

I will hopefully not get too technical here but there are some important points raised in the paper which are perfectly testable in an autism research and other context.

So:

• The proposed model is based on two different rat urinary phenotypes distinguished by a favourite metabolomics method, NMR spectroscopy - one of the gold standards. One rat phenotype - the 'HIP' phenotype - produces rather a lot of urinary hippurate, whilst the other phenotype - the chlorogenic acid phenotype - is characterised by low levels of urinary hippurate and elevated levels of 3-(3-hydroxyphenyl)propionic acid (3-HPPA). The differences between the biochemistry of the two phenotypes is gut bacterial composition not readily attributed to factors like different diets.
• Proposals for the bacterial agents and biochemical pathways pertinent to these rat phenotypes is given with specific focus on the origin of the benzoic acid that consequently forms the hippurate levels seen in the HIP phenotype. Words like cinnamic acid and PAL are also included.
• Transposing the various biological reactions noted in phenylalanine and tyrosine on to tryptophan, Clayton moves through the production of IAG from tryptophan via a most interesting intermediary compound called indole-3-acrylic acid (IAA). IAA is thought to be quite a reactive molecule which subsequently conjugates with glycine to form IAG, also able to potentially do various things because of its flat planar chemical geometry. There is for example, some prior speculation that IAA may have some 'membrane-busting' potential in relation to autism as a result of its dose-dependent effect on prostaglandin (E) production, but this observation still remains preliminary. 

Acknowledging that this is a hypothesis paper and so still subject to some degree of testing, I am very curious about some of the concepts detailed. Elevated levels of 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) have been reported in cases of autism, a compound which fits rather nicely into the whole phenylalanine-bacteria connection with particular focus on an old bacterial friend, Clostridia. This accompanied by various other related findings in cases of autism such as elevated levels of urinary 4-hydroxyhippuric acid detailed in this paper adds to the curiosity.

Speculation on some role for IAG and precursors in relation to autism and particularly some reported [non-significant] differences to levels according to use of a gluten-free diet, has again been something of interest to me for a while. Allowing for interfering variables based on urine sample concentration noted in some cases of autism, the question of why a gluten-free diet in particular, might affect urinary IAG levels has always been a puzzle. Does a gluten-free diet affect gut bacterial composition? Likely, according to this paper. Does the gluten-free diet affect gut permeability? Yes it probably does to some extent and the de Magistris results seem to back that up in cases of autism. Could gut bacteria contribute to gut permeability either directly or peripherally through the formation of IAG or related mechanisms? Mmm... I dunno.

There is a lot to take in with this recent paper and even after a few reads I still find myself not fully understanding all of the chemistry. Nevertheless an important connection has been posited between our gut bacterial masters and their potential effect on important aspects of our biochemistry. I would like to think that I will post further about some of the implications of this hypothesis as more research starts to move into this area.

To end, an oldie but goodie which was the soundtrack to a favourite film of mine. Cue Richard Dreyfuss.. "I never had friends later on like the ones I had when I was 12...".

* Clayton TA. Metabolic differences underlying two distinct rat urinary phenotypes, a putative role for gut microbial metabolism of phenylalanine and a possible connection to autism. FEBS Letters. February 2012.
DOI: 10.1016/j.febslet.2012.01.04