The prebiotic galactooligosaccharide (B-GOS) and autism: just add to poo(p)

Yes, it is childish but...

With all the continued chatter on a possible role for the collected gut microbiota - those wee beasties that inhabit our deepest, darkest recesses - in relation to some autism (see here for example), the paper by Roberta Grimaldi and colleagues [1] (open-access available here) provides yet more potentially important information.

So, poo(p) samples were the starring material in the paper - "obtained from three non-autistic children and three autistic child donors"- and specifically what happened when something called B-GOS "a prebiotic galactooligosaccharide" was added to samples following their journey through a "Three stage continuous culture gut model system" otherwise known as an artificial gut. Said gut model based at Reading University has already been the topic of other news (see here).

As well as looking at the initial bacterial profile of those stool samples, researchers plotted the changes to the stool's inhabitants (or what was left of the stool) over the course of B-GOS addition, as well as looking at things like the "production of SCFAs [short-chain fatty acids] in the fermentations" and other metabolites via the gold-standard chemical analytical technique called 1H-NMR (see here for more details).

Results: "Consistent with previous studies, the microbiota of ASD [autism spectrum disorder] children contained a higher number of Clostridium spp. and a lower number of bifidobacteria compared to non-autistic children." With the addition of B-GOS to the 'mixture', researchers reported on a significant increase in bifidobacterial populations at the different stages of their gut model and in samples from both those with autism and those without autism. Such "bifidogenic properties of B-GOS" are not unheard of.

As to the metabolites of those bacteria present in the poo(p) samples, there were some interesting knock-on effects noted in both raw and B-GOS supplemented samples. "Our data show a lower concentration of butyrate and propionate in autistic models, compared to non-autistic models, but no
differences in acetate before adding B-GOS into the system." Propionic acid (propionate) has some research history with autism in mind (see here). Butyric acid (butyrate) is something of a rising star in quite a few domains, having also been mentioned in the context of autism too (see here). Indeed it's interesting to note that B-GOS administration "mediated significant production of... butyrate... simulating the transverse and distal colon respectively. There was no effect on propionate." The findings of lower starting levels of butyrate in samples from children with autism were also substantiated by the NMR analyses undertaken. Increases in butyrate and changes to various other metabolites ("increasing ethanol, lactate, acetate and butyrate and decreasing propionate and trimethylamine") were also noted via this analytical method for this group.

A long quote coming up: "This in vitro study showed promising and positive results in that supplementing the microbiota of ASD children with 65%B-GOS may manipulate the gut bacterial population and alter metabolic activity towards a configuration that might represent a health benefit to the host. However, further work will be required to assess such changes in an in vivo human intervention study."

Just before anyone makes a run on B-GOS or any similar product however, I do need to stress a few important points. First, this was a study of poo(p) samples from 3 autistic children compared with samples from 3 non-autistic children. Aside from the small participant numbers, we don't know anything about participants' various comorbidities (although we know they were "free of any metabolic and gastrointestinal diseases") and only limited information on their dietary habits and medication history. Second, poo(p) was the target material included for analysis and what happened when B-GOS was supplemented during the journey through the artificial gut model. This study said nothing about what happens when real people with autism take B-GOS orally for example, and how it might affect gut bacterial populations and metabolites as it progresses down a real gastrointestinal (GI) tract. This also includes a lack of information on any potential side-effects in a real-world situation. We are also assuming that any supplement survives the stomach. There is quite a bit more to do in this area.

But for now, I stick to the idea that the Grimaldi paper provides some potentially important information and certainly, some new routes/methods for further study of the link between prebiotics, probiotics and synbiotics in the context of the gut microbiota and autism...

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[1] Grimaldi R. et al. In vitro fermentation of B-GOS: Impact on faecal bacterial populations and metabolic activity in autistic and non-autistic children. FEMS Microbiol Ecol. 2016 Nov 16. pii: fiw233.

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Grimaldi R, Cela D, Swann JR, Vulevic J, Gibson GR, Tzortzis G, & Costabile A (2016). In vitro fermentation of B-GOS: Impact on faecal bacterial populations and metabolic activity in autistic and non-autistic children. FEMS microbiology ecology PMID: 27856622

Acyl-carnitine profiles and autism

No doubt alongside quite a few others, I was interested to read the latest paper from Richard Frye and colleagues* (open-access) discussing the potential links between an animal model of autism spectrum disorder (ASD) and real-world ASD in a particular cohort of participants.

This is not the first time that I've talked about (a) the work of Dr Frye - as per my [don't panic] post on folate receptor autoantibodies in cases of autism (see here) and (b) some of the difficulties attached to translating rat/mouse model findings in autism research into 'real-world' findings (see here). Indeed this last point might also tie into my recent musings on the use of LPS in autism research (see here).

Daddy o' microbiology @ Wikipedia  

My interest in the latest Frye paper was further piqued upon realising that Derrick MacFabe was also part of the authorship team given his insightful work on how administration of propionic acid - a short chain fatty acid - to rodents might provide some interesting perspectives overlapping with various findings noted in cases of autism (see my previous post here).

The Frye paper is open-access and available to all but here are a few points worth mentioning:

• Based on the animal model work looking at propionic acid (PPA) infusions and some of the collected effects (presentation of autism-type behaviours**, mitochondrial issues***, abnormal measures of glutathione, etc.), the aim of the current study was to map out whether some of the observations in the PPA rodent model were also present in real-life autism.
• Based on a clinic-based sample of children with autism (N=326), acyl-carnitine panels were conducted on about two-thirds of participants (n=213).
• Acyl-carnitines, as their name suggests, are related to carnitine which has previously been tied back to autism (see here) and represent a number of different compounds - complexes of carnitine and various fatty acids - involved in the transport of fatty acids into the mitochondrial matrix.
• The previous PPA rodent model indicated some disturbance in the amount of various acyl-carnitines, particularly with regards to short- and long-chain fatty acids (not so much in the medium-chain fatty acids) reflective of some mitochondrial dysfunction.
• So the authors looked at short-, medium- and long-chain acyl-carnitines in their autism cohort to see if there were any overlaps compared with the PPA treated animals.
• Results: 35% of the autism cohort showed "an increase in three or more acyl-carnitines when initially measured". When further testing were conducted on some of these participants showing elevation, this figure was revised to 17% of the cohort who "demonstrated consistent elevations in short-chain and long-chain, but not medium-chain, acyl-carnitines". You'll note the similarity with PPA animal model in terms of the short- and long-chain acyl-carnitines.
• Furthermore, four participants were also examined with regards to glutathione and oxidative stress markers. As per the quite consistent literature on things like total- and free-reduced glutathione (see this post), compared with controls, there were some familiar trends emerging.

And relax.

There is a lot to take in from this paper both in the protocol and testing undertaken and the possible interpretation of findings. A quote best sums up the results: "This study has demonstrated that ~17% of children with ASD manifest biomarkers of abnormal mitochondrial fatty-acid metabolism that parallel similar biomarkers in the PPA rodent model of ASD".

As perhaps expected, there has been some press attention following the publication of this paper. The headline: Researchers discover link between certain types of autism and gut bacteria has been a common one, reflective of the fact that when it comes to the production of PPA outside of injecting the stuff directly into the rodent brain, the gut and in particular, certain types of gut bacteria, have been suggested as a route to PPA production in cases of autism. Indeed, I'm taken back to the findings by Wang and colleagues from Oz (see this post) on levels of fecal short chain fatty acids (SCFAs) in their cohort of children with autism which included propionic acid (propionate). I'm also reliably informed that there is 'more to come' from the Australian research group in the coming months.

There are a few final aspects to the Frye study which I should have mentioned earlier. This includes the notion that "it is very likely that MD [mitochondrial dysfunction] is acquired" given that in the most part, both nuclear and mitochondrial DNA examinations in their cohort were negative for anything that might genetically account for the results. I'll be coming to DNA and mitochondrial issues in a subsequent 'training' post scheduled quite soon but one can perhaps see how this might strengthen any argument of disruptions to gut bacteria/microflora facilitating the production of elevated PPA onwards to disrupting acyl-carnitine chemistry.

Additionally there is the implication based on the PPA rodent model that affecting PPA production could be a potential therapeutic route for some cases of autism at some point. I don't really want to go too far into the hows and whys of this suggestion at the current time given the current lack of evidence for any effect. Aside that is, from drawing your attention to a related field of inquiry into how disrupting PPA-producing gut bacteria might have some important implications for those suffering from conditions like propionic acidaemia****.

I'm not going to get too carried away with the Frye results as they stand. As per my previous post on PPA, there is still a bit of a stretch from injecting rodent brains with PPA and recording outcomes to suggesting that gut bacteria will be able to produce enough PPA so as to exert a similar effect in cases of autism. At least one group is asking the same question***** (open-access). That being said, I am still interested in the details of this study and how the authors have at least tried to model animal findings into real-life autism. Indeed how we really should be putting a lot more research effort into looking at mitochondria and autism as we potentially also should gut bacteria and autism.

A song to finish... something mellow yet catchy.... Lovefool by the Cardigans.

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* Frye RE. et al. Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder. Translational Psychiatry. 2013; 3: e220.

** MacFabe DF. et al. Effects of the enteric bacterial metabolic product propionic acid on object-directed behavior, social behavior, cognition, and neuroinflammation in adolescent rats: Relevance to autism spectrum disorder. Behav Brain Res. 2011; 217: 47-54.

*** Thomas RH. et al. Altered brain phospholipid and acylcarnitine profiles in propionic acid infused rodents: further development of a potential model of autism spectrum disorders. J Neurochem. 2010; 113: 515-529.

**** Mellon AF. et al. Effect of oral antibiotics on intestinal production of propionic acid. Arch Dis Child 2000; 82: 169-172.

***** El-Ansary AK. et al. Comparative study on the protective effect of carnosine and carnitine against pro-inflammatory/pro-oxidant effects of clindamycin and propionic acid administrations to hamsters. African Journal of Microbiology Research. 2013; 7: 103-114.

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Frye RE, Melnyk S, & Macfabe DF (2013). Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder. Translational psychiatry, 3 PMID: 23340503

Propionic acid and autism

Another post, another organic acid (!) but please don't click away just yet as I attempt once again not to blind you with science. The topic of the day today is propionic acid (PPA) otherwise known as propanoic acid and the very tentative suggestions of some link to autism. 

Search on-line for propionic acid and you're quite likely to get lots of industrial chemical information about it including a materials safety data sheet (MSDS) (here) implying that this stuff is toxic, combustible and corrosive. So why on earth am I talking about it with regards to autism?

Well, like many things in modern life, the chemical industry isn't the only source of 'chemicals' as per our wonderful chemical factory that is the human body and the surprising effects of some of the bacteria which inhabit us. Not for the first time, I might add, has bacteria and biochemistry come together on this blog (see here) and probably not the last time either.

PPA is an organic acid - a short chain fatty acid - which as well as being used as a food additive / preservative and used in the process of polymer production apparently, is also what might be considered a natural product as a result of its endogenous production. Short chain fatty acid you say? Y'mean like the ones discussed not so long ago in relation to autism by Wang and colleagues? Yes, the very same.

Continuing with autism in mind, I am taken back to this news report from 2007 where Dr Derrick MacFabe discussed this piece of research* (followed up by this study** too) suggesting that you are what you eat in terms of foodstuffs and bacteria combining to produce PPA and what effect it had on the rat brain following "intraventricular infusions" (to you and me injecting the stuff directly into rat brains). Dr MacFabe has very definitely taken the lead when it comes to looking at PPA and autism. 

Indeed I was interested to see the effects of PPA administration on rat brains including activated microglia and decreased glutathione alongside the more overt effects on rat behaviour. Understanding that rats (like mice and fish) are not humans, indications of issues with glutathione have been quite strongly shown to be related to cases of autism and indeed the brain has become a recent focus for this area of research. As for microglia, I would once again refer you to this post by Paul Patterson on the suggested dietary habits of microglia in autism with the promise of a post on this research area soon.

Other research groups have also entertained a possible role for PPA in autism. The paper by El-Ansary and colleagues*** replicated the results from the MacFabe group and indeed added a few new aspects also making mention of lactate dehydrogenase and elevations in IL-6 among other things; both factors having been reported in connection to autism (here and here respectively). I think we can, with some degree of confidence, assume from this collected work, that propionic acid injections are probably not good news for rats.

We move onwards then to another piece of research by El-Ansary and colleague**** (full-text) looking at the possibility that supplementation with omega-3 fatty acids might afford some protection against the effects of PPA administration to rats. They reported that omega-3 supplementation did indeed alter some of the parameters associated with PPA administration including some interesting data on caspase-3, an executioner molecule involved in apoptosis previously reported in cases of autism (see here for more information). It seems rats fed a diet rich in omega-3 fatty acids might have some protection against various effects of PPA administration. I assume this ties into this paper by Thomas and colleagues***** (including Dr MacFabe).

So to summarise. An overlap between direct PPA administration and some key biochemical changes noted in cases of autism. Fine. A potential protective effect via use of omega-3 fatty supplementation from PPA administration. OK (fatty acids have some history in cases of autism as per this older post). PPA found to be elevated in cases of autism (based on the analysis of stools). Right.

Reading all that might convince some people of a role for PPA in autism but I'm going to play devil's advocate for a second and suggest why we need more research in this area. First, as mentioned rats aren't human beings. Rats might make good models for human being - as do some mice - but rats are not human beings. Second, administration of PPA was either intracerebroventricularly or at "neurotoxic doses" over 3 days (250mg/kg body weight). Either of these two factors whilst good for studying rats in a lab over a relatively short space of time is probably not going to be representative of chronic PPA exposure in people with autism. Unless of course that is, that something causes a huge acute surge in PPA that is. Finally(!) aside from the Wang findings on fecal PPA levels, the research in this area is still a little bit scant in terms of PPA levels in other biofluids. I haven't for example, seen anything as of yet looking at whether rates of the metabolic disorder propionic acidaemia is any more prevalent in cases of autism than not autism or likewise whether propionic acidaemia is accompanied by an increase in cases of autism accepting the seriousness and poor prognosis of this rare metabolic conditions. A few possible routes to further study perhaps.

That being said, I am still intrigued about propionic acid and the research done so far with regards to autism whether animal model-based or direct measurement. The whole connection back to short chain fatty acids, digestion and gut bacteria is an interesting and very under-researched area with autism in mind -  indeed with lots of other behavioural / cognitive / psychiatric conditions in mind. Could it hurt to put a little more research efforts into this area?

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* MacFabe DF. et al. Neurobiological effects of intraventricular propionic acid in rats: possible role of short chain fatty acids on the pathogenesis and characteristics of autism spectrum disorders. Behavioral Brain Research. 2007; 176: 149-169.

** MacFabe DF. et al. Effects of the enteric bacterial metabolic product propionic acid on object-directed behavior, social behavior, cognition, and neuroinflammation in adolescent rats: Relevance to autism spectrum disorder. Behavioral Brain Research. 2011; 217: 47-54.

*** El-Ansary AK. et al. Etiology of autistic features: the persisting neurotoxic effects of propionic acid. Journal of Neuroinflammation. April 2012.
DOI: 10.1186/1742-2094-9-74

**** El-Ansary AK. et al. On the protective effect of omega-3 against propionic acid-induced neurotoxicity in rat pups. Lipids in Health & Disease. 2011; 10: 142.

***** Thomas RH. et al. The enteric bacterial metabolite propionic acid alters brain and plasma phospholipid molecular species: further development of a rodent model of autism spectrum disorders. Journal of Neuroinflammation. 2012; 9: 153.

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Short chain fatty acids and ammonia in autism

It is perhaps apt that Jon Brock over at Cracking the Enigma recently posted about the Australasian Society for Autism Research highlighting the various links being forged regarding autism research in that part of the world. Apt because there are several very good autism research groups producing findings which have some potential real-world application to autism, in that part of the World. I speak in particular of the team based at the Sansom Institute for Health Research based at the University of South Australia. Members of UNISA recently publishing a fine review of how glutathione and relations show quite a consistent relationship to cases of autism. Autism research movers and shakers please take note.

Indeed another paper published by Wang and colleagues* adds to the repertoire, suggesting that levels of fecal short chain fatty acids (SCFAs) and ammonia were higher in their cohort of children with autism compared to asymptomatic controls and hence showing alteration in the fermentation process.

As a sort of introduction to SCFAs and fermentation, readers might find this article by Topping & Clifton** (full-text) and this article by Macfarlane & Macfarlane*** (full-text) to be of interest. Basically, think carbohydrates and gut bacteria and eventually you head the direction of SCFAs. If you happen to brew your own beer, fermentation might not necessarily be a strange concept alongside memories of 'auto-brewery syndrome' (not personal memories I might add).

Unfortunately I can't post a link to the full-text but here are a few details:

• Based on the collective studies of Dr Wang's PhD (scroll down to see her thesis details), this study looked at fecal specimens from a relatively small group: 23 children (mean age ~ 10 years) diagnosed with ASD compared with 31 age-matched controls.
• Although not everyone's choice of biological medium to work with, fecal samples - as in poo samples - were analysed via various methods including colorimetry (ammonia) and gas chromatography (SCFAs). Other compounds such as p-cresol were analysed via liquid chromatography.
• Results: fecal concentrations of total SCFAs were elevated in autism samples (p=0.012) as were levels of ammonia (p=0.007). Interestingly no elevation was observed in levels of p-cresol in autism samples as has previously been described. 
• More specifically with individual SCFAs, some interesting compounds cropped up as being elevated in autism samples including: propionic acid, butyric and isobutyric acids and valeric and isovaleric acids. Indeed out of the battery of SCFAs analysed, only caproic acid showed no significant difference between the groups.
• The authors conclude that fecal levels of these large bowel fermentation products might tie into the accompanying gastrointestinal (GI) findings observed in some children with autism and differences in the bacteria inhabiting the darkest recesses.

There are some obvious caveats to accompany this latest study. Aside from participant numbers being relatively small and covering quite a lot of chronological age in childhood, and the slight discrepancy in controlling for gender, there was quite a bit of 'overlap' when it came to individual results from the autism and control groups. Indeed if I were to criticise at all, I perhaps would have like to have seen some plotted information on the dispersion of values obtained from individuals just to visualise and ensure that results were not skewed by a few outlier results. As has however been discussed recently, outliers in a heterogeneous condition like autism are very often a source of great interest.

This is not the first time that the words 'autism' and 'fermentation' have come to my attention. Quite a few years back I remember some interesting work being done at Cranfield University by Maria Pilar Bilbao Montoya as part of her higher studies. Working with gastroenterologist, John Hunter, Maria presented some initial work suggesting similar things in their cohort I believe. I might be wrong but I also remember that Dr Sophie Rosseneu might have had some unpublished work in this area also as a tie up with her findings on yeasts and autism?

Memories aside, there are some interesting points to take from this research. SCFAs such as propionic acid (PA) have recently been seen in the autism research arena as per this paper (full-text) by El-Ansary and colleagues looking at what happens when you inject rats with PA in sizeable quantities. For those of you who are unimpressed with the proposed PA-gut-brain link, maybe a few other choice papers might be useful, here and here. And perhaps some evidence for the presence of PA in other biological fluids from cases of autism?

I've talked ammonia before on this blog with reference to some mitochondrial findings. Wang and colleagues look more to the GI effects of ammonia given that it was detected in stool samples and how this might tie into issues with gut permeability so commonly discussed. Indeed ammonia - the whole nitrogen balance - takes us back to some old friends, glutamine and glutamate, one of which has been making quite a bit of news lately (here's the paper abstract).

The big question has to be why the reported differences in this latest study? I can't give a specific answer to this question but given the interest in carbohydrate metabolism and autism, the suggestion of things like lactose intolerance, added to the growing research base on gut bacteria and autism, one has to suspect that the answer might lie, partially at least, somewhere in these regions.

* Wang L. et al. Elevated fecal short chain fatty acids and ammonia concentrations in children with autism spectrum disorder. Digestive Diseases and Sciences. April 2012
DOI: 10.1007/s10620-01202167-7

** Topping DL. & Clifton PM. Short-Chain Fatty Acids and Human Colonic Function: Roles of Resistant Starch and Nonstarch Polysaccharides. Physiological Reviews. 2001; 81: 1031-1064

*** Macfarlane S. & Macfarlane GT. Regulation of short chain fatty acid production. Proceedings of the Nutrition Society. 2003; 62: 67-72