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

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

That recent metabolomics study on autism (part 2)

Consider this a part-two to my previous post introducing the paper from Dr Xue Ming and colleagues* on metabolomics, amino acids and gut bacteria with autism in mind. It's going to be another micropost (sort of!) given that quite a bit of the background has already been discussed previously.

So straight to the paper:

• As expected, the study from Yap and colleagues** (including Prof. Nicholson) was the template for undertaking this study.
• Spot urine samples were collected from 48 children diagnosed with DSM-IV autism; in some cases confirmed by the gold-standards that are ADOS and ADI, and compared with 53 matched asymptomatic controls.
• Alongside the various screening measures, a gastrointestinal [disorder] GI severity scale was used to ascertain the presence of GI dysfunction. (verified in 29/48 children with autism).
• Mass spectrometric analysis of the urine sample, blah, blah, blah, and out of a total of 391 metabolites, confirmed metabolites, located in samples, 82 of them were altered between autism and control samples.
• Amino acids et al: several amino acids were significantly lower in the autism group including glycine, serine and the alanines. Taurine also featured. Indeed quite a few of the gamma glutamyl amino acids were also reduced in the autism group with a link to GI disorders potentially suggestive of issues with gamma glutamyl transpeptidase.
• Gut bacterial metabolites: some really interesting stuff here and very much influenced by the presence of GI problems or not. Of particular note, "significantly increased levels of 2-(4-hydroxyphenyl)propionate and taurocholenate sulfate" and reduced levels of 3-(3-hydroxyphenyl)propionate and 5-aminovalerate.
• Mention is also made of some of the gut bacterial findings previously made with autism in mind, and even the possibility of gut hyperpermeability (leaky gut) as influencing the absorption of metabolites. Think back to Paul Patterson' recent announcement on mice, leaky gut and gut bacteria.

A couple of quick observations. Taurine: OK, more an organic acid than an amino acid. Found to be significantly lower in the autism group results compared with controls in this study, but by contrast, elevated in the Yap study. One could argue that there were participant geographical differences (Ming: USA vs. Yap: Australian/Swiss) which might reflect genetic, dietary or environmental differences across different geographical groups. It's interesting to note also that in the Yap paper they noted that "taurine concentrations were hypervariable in the autism group". That and the differences across analytical technologies (Ming: mass spec vs. Yap: NMR). Autisms not autism? Who knows?

Lower urinary glycine and indeed N-acetylglycine were also picked up in the autism group of the Ming study. I don't want to make too much of this at the moment but perhaps will throw in two possibly relevant things: (a) that very interesting paper from Andrew Clayton on autism, aromatic amino acids and gut bacteria (see this post) which talked about benzoic acid and hippuric acid and the link with glycine and (b) the very interesting area of glycine and sleep (see this post). I'm not making any value judgements bearing in mind the focus on urinary glycine not plasma levels of glycine, so just putting it out there.

There's not too much more to say on the the Ming paper apart from being a really interesting piece of research and with a strong requirement for independent scientific replication. That and given the focus on comorbid GI conditions appearing alongside some cases of autism, perhaps a lot more inspection into the root causes and management of them. Indeed look no further than the special Pediatrics supplement on autism for quite an interesting opinion paper*** on this topic with some pretty big hitters as part of the authorship group.

And rest.

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* Ming X. et al. Metabolic perturbance in autism spectrum disorders: a metabolomics study. J Proteome Res. October 2012.

** Yap IK. et al. Urinary metabolic phenotyping differentiates children with autism from their unaffected siblings and age-matched controls. J Proteome Res. 2010; 9: 2996-3004.

*** Coury DL. et al. Gastrointestinal conditions in children with autism spectrum disorder: developing a research agenda. Pediatrics. 2012; 130: S160-S168.

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Ming X, Stein TP, Barnes V, Rhodes N, & Guo L (2012). Metabolic Perturbance in Autism Spectrum Disorders: A Metabolomics Study. Journal of proteome research PMID: 23106572

Inborn errors of creatine metabolism and autism

I might have said it before but inborn errors of metabolism represent a particularly interesting group of conditions relevant to this blog. I have tended to focus on one particular metabolic condition, phenylketonuria (PKU), simply because it represents the archetypal 'diet-can-affect-behaviour-and-cognition' condition. That and the fact that PKU has been known to overlap with certain cases of autism.

In this post I turn my attention to another group of metabolic disorders related to creatine and its biosynthesis and metabolism. The papers that brought me to this post are this one from Chilosi and colleagues* (full-text) and this one from Kurosawa and colleagues** (full-text).

In brief, Chilosi et al describe how supplementation with the amino acid L-arginine (one of the precursor compounds used in the formation of creatine) might be able to positively affect some of the symptoms associated with creatine transporter deficiency. Kurosawa et al reported results following supplementation with cyclocreatine (CincY) in a mouse model of creatine transporter deficiency. I might add that I am not endorsing these or any other course of action without the proper medical advice, merely describing what was published.

Reversing a little, creatine (not to be confused with a relation, creatinine, something else that has cropped up on this blog - here and here) is probably best described as an energy supplier. A fusion of the amino acids L-arginine and glycine form guanidinoacetate which then goes on to creatine. Creatine continues onwards on to prop up production of ATP. It's obviously a little more complicated than just that but I'm not here to deliver a biochemistry lesson.

Inborn errors of creatine metabolism normally fall into one of a few categories best described here. You'll note that one of the conditions is something called X-linked SLC6A8 creatine-transporter deficiency.  Just keep that in mind for now; that and the words 'autistic-like' with reference to the behavioural presentation.

So what about with autism in mind:

• Well, let just say that inborn errors of creatine metabolism are probably not going to be very common in autism as per this review by Schiff and colleagues*** (full-text) and this paper by a familiar name, Wang and colleagues**** (of SCFAs fame). That being said, I don't know if screening for creatine metabolism issues in cases of autism is widespread or not.
• Bearing in mind the important energy function of creatine, in turns out that getting creatine into cells via the SLC6A8 gene is quite prevalent in certain areas of the body particularly the brain. Indeed there is some suggestion that moving from monkeys to humans might have had a lot to do with this and another gene and their transporting ability as per this study by Pfefferle and colleagues***** (full-text). With all this creatine floating around the grey stuff (well, the pink-red stuff), it is perhaps not surprising that a few imaging studies have looked at brain creatine distribution in cases of autism. Very much like the use of creatinine as a ratio marker for quantifying urinary metabolites, so creatine levels serve a similar function when it comes to the use of 1H magnetic resonance (MR) spectroscopy to quantify various brain metabolites. Much also like the suggestion of issues with the use of the ratio marker creatinine (see here) in autism so brain creatine levels also seem to be suggestive of certain 'findings' in various brain areas (see here, here and here).
• Remember SLC6A8 and the 'X-linked' issue? Well, there has been some interesting research done there too with autism in mind (here, here and here). X-linked refers to the chromosomal make-up of boys and girls, such that girls have two X chromosomes (XX) and boys have two distinct chromosomes (XY). I should also make mention other conditions such as Klinefelter's syndrome where an extra X chromosome is present. The male bias noted in autism has led some people to speculate on some role for the X chromosome in relation to the condition, as detailed in this paper by Marco & Skuse****** (full-text). 

There are some interesting points to take from this collected research on creatine in relation to autism. Probably first and foremost is the further evidence for autism being a very complicated condition with seemingly lots of paths to the presentation of behaviours gathered under the autism label. I know many people won't find any new revelations in this statement but issues with creatine metabolism is a good example.

Second is a reiteration of the importance of the inborn errors of metabolism to concepts like autism. OK so these conditions are likely not going to be universal to all people with autism (as far as we know); but for a minority they might be, and as per the PKU connection and recent Chilosi / Kurosawa findings, there may very well be interventions which might impact upon behavioural presentation.

Finally, screening. I could be here all day reciting what screening 'could' be undertaken when a diagnosis of autism is received. I won't aside from mentioning things related to SPAD, PFIES, coeliac disease, inflammatory markers, antioxidant markers, iron levels, zinc levels, etc, etc. Assuming learning disability is also present alongside the presentation of autism, screening for errors in creatine metabolism might also be a possible addition to that list.

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* Chilosi A. et al. Neuropsychological profile and clinical effects of arginine treatment in children with creatine transport deficiency. Orphanet Journal of Rare Diseases. June 2012.
DOI: 10.1186/1750-1172-7-43

** Kurosawa Y. et al. Cyclocreatine treatment improves cognition in mice with creatine transporter deficiency. The Journal of Clinical Investigation. July 2012.
DOI: 10.1172/JCI59373

*** Schiff M. et al. Should metabolic diseases be systematically screened in nonsyndromic autism spectrum disorders? PLoS ONE. 2011; 6: e21932.

**** Wang L. et al. Is there a role for routinely screening children with autism spectrum disorder for creatine deficiency syndrome? Autism Research. 2010; 3: 268-272.

***** Pfefferle AD. et al. Comparative expression analysis of the phosphocreatine circuit in extant primates: Implications for human brain evolution. Journal of Human Evolution. 2011; 60: 205-212.

****** Marco EJ. & Skuse DH. Autism-lessons from the X chromosome. Social Cognitive & Affective Neuroscience. 2006; 1: 183-193.

Mercury, creatinine, outliers and autism

There are certain areas of the autism research landscape which have created real divisions among some professionals, scientists and people with autism and their families. One of them surrounds the relative contribution (or not) of heavy metals such as mercury to the onset and presentation of autism in some people. The theory being that environmental exposure to mercury (in whatever form) may somehow interfere with things like neuronal development and contribute to some of the symptoms seen in autism at least in some people. Quite a good overview of the whole debate is presented by Garrecht & Austin* (full-text). The obvious background support to this theory are the very real effects that have been documented as being associated with environmental mercury exposure, particularly in children. The other side of the coin is the epidemiological evidence suggestive of no overall connection between certain mercury-related exposure factors and events and the rising numbers of cases of autism.

A recent study by Barry Wright and colleagues* (full-text) contributes some more to this area of research with their findings of no overall difference in urinary mercury excretion, but (and it is a big but) a small number of outlier results perhaps warranting further research attention.

So, their study summarised:

• From an initial participant group (N=251) comprising 54 UK children with autism, 42 of their siblings and 155 children without autism (121 going to mainstream school and 34 children attending a special school), urinary mercury levels were attained in 230 children via ICP-MS. The analysing laboratory was blind to who was who.
• Based on analysis of mercury concentration either correcting or not correcting for creatinine (an important urinary ratio marker), no significant group differences were noted across the participants. Even allowing for age, gender and the number of amalgam fillings, there were no significant differences and including analysis of various other metals such as lead, cadmium and copper, no significant group differences were noted.
• That being said, there were a small number of 'outliers' whose results appeared to indicate elevated levels of mercury to be present. These results appear in the autism and special school group (assuming that this group included children with other learning or developmental issues).

Depending on your viewpoint, you could interpret these results a number of ways. You could say, no overall group differences so mercury is not going to be an issue in relation to autism and is certainly not driving the increase in cases. On the other hand you could say, OK no group effect but given that autism is a heterogeneous condition driven by the phrase 'if you've met one person with autism, you've met one person with autism' the results don't preclude a small subgroup whose presentation might be tied into a factor like mercury. It all depends on your point of view. Also as per the study discussion, levels of urinary mercury is one thing, levels of mercury in blood, plasma, brain, etc. is another.

I also note another interesting detail from the study. Let me run a sentence past you: "A lower creatinine in the ASD group (not statistically significant) raises the mercury to creatinine ratio in this subgroup, but not to statistically significant levels". I was interested to read this in view of my own research interest in creatinine in relation to autism (see here). The logic goes that a lower level of urinary creatinine might artificially inflate whatever other metabolite you are looking at as per this previous post. Granted in this study, they looked at both corrected and uncorrected concentrations and found no group difference on either occasion but still the question of why creatinine levels might have been lower is an interesting one.

* Garrecht M. & Austin DW. The plausibility of a role for mercury in the etiology of autism: a cellular perspective. Toxicological & Environmental Chemistry. 2011; 93: 1251-1273
DOI:  10.1080/02772248.2011.580588

** Wright B. et al. A Comparison of Urinary Mercury between Children with Autism Spectrum Disorders and Control Children. PLoS ONE. February 2012.
DOI: 10.1371/journal.pone.0029547

Specific Gravity vs creatinine for urinary correction

Apologies if you tuned in for a post about autism, but this is another 'other musings' entry (although I might mention autism at some point). Please, stay with me (this is not an April Fool) because I hope it might be of some interest; if anything else it might make for a few factoids vis-a-vie Steve Wright round the water-cooler at work the next day.

What's to say about urine? A waste product that we all produce in copious amounts throughout our lifetime. Constitution-wise it contains quite a bit; a good idea of what and how much is shown here. More recently urine, or rather the analysis of urine has been central to a relatively new branch of biological chemistry called metabolomics - studying the chemical 'fingerprints' left behind by biological processes.

The theory is that the pattern of our waste products in urine for example, whilst being unique to each individual, contains enough information for science to start looking at groupings based on compounds or levels of compounds excreted, which may eventually lead to predictions on our risk of disease or alternate biological markers for particular conditions or diseases.

Don't believe me? Well have a look at these abstracts suggestive of urine markers for kidney cancer, coeliac disease and depression to name but a few.

Last year there was quite a bit of excitement when a team based in London published this paper suggestive of potential urine biomarkers for autism. Accepting a few criticisms of the paper, some of the markers indicated for autism samples such as taurine, glutamate and various potential gut dysbiotic markers have been discussed previously as being possibly relevant, in various biological mediums.

Any more convinced? Yes... No...

OK, back to urine. One of the many differences, both intra- and inter-person, with regards to urine is concentration; that is, how concentrated or dilute our urine is. Urine concentration is affected by many different things; how hydrated our body is, whether we have engaged in any exercise, etc. Concentration of urine is important in working out the total amount of a compound or set of compounds also. If we imagine that a concentrated urine sample will contain higher levels of compounds quantitatively than a more dilute sample, you can see how sample concentration might affect studies looking at quantitative urinary biomarkers for a specific condition.

There are various ways of 'correcting' for urine concentration/dilution. Probably the most widely used is to measure urinary creatinine and correct sample concentration against creatinine. Whilst not wishing to provide Biochemistry 101 for creatinine metabolism, it is important at this point to make a distinction between creatinine and a related compound, creatine (although the two are connected).

So then normally when someone is trying to quantify one or more compounds in urine they may say something like: 100mg compound X / g/l creatinine. This means that 100 miligrams of compound X was detected per gram per litre of creatinine. (There are other units of concentration also used but I will not complicate things).

Creatinine is not alone as a marker for urine sample concentration (spot samples or 24-hour samples). There are others. One measure is the specific gravity (SG) of the sample. Anyone who has an interest in home brewing beer might know something about SG. SG is the ratio of the density of a sample relative to the density of water, assuming equal temperature and pressure. Pure water under set conditions has an SG of 1. A urine sample with all its dissolved compounds should have an SG greater than 1. Before you look away, this is as technical as I am getting.

Measuring the SG of urine is a tried and tested method and, in many respects, an easier option than assaying for creatinine (which depending on your method of analysis can include some quite 'explosive' steps). What's the difference between creatinine levels and SG I thought I heard you say...?

Well, there shouldn't be too much. There has been a long running debate about whether creatinine is a better marker of concentration over SG and vice-versa. The light at the end of the tunnel seems to suggest that there is little or no difference between the two measures when applied to the general population.

Fine, end of story. Not quite. There has been, for quite a few years now, some initial evidence to suggest that levels of creatinine in autism for example, are perhaps slightly aberrant compared to not-autism. Not trying to be a blatant self-publicist here, but I put my hand up because my colleagues and I have published on this a few years back. More recently also, similar findings have been reported by other authors; although I stress that this is not without some controversy.

Why might creatinine levels be unusual in autism? I dunno - potentially lots of reasons ranging from fluid intake to activity levels through to how much meat there is in the diet. What I do know is that if creatinine levels are somehow more consistently perturbed (lower), using it as a concentration corrector might, I stress might, indicate that comparator compounds are present in higher levels than they really are. I will stop there.

Next time you pop to the WC for a sprinkle, take a moment to think about what exactly is going into the bowl and how science stands to gain so much from it.