A test "that distinguishes ASD fast response constipation from ASD persistent right-sided constipation"?

The quote heading this post - "A test that distinguishes ASD [autism spectrum disorder] fast response constipation from ASD persistent right-sided constipation" - comes from the findings reported by Stephen Walker and colleagues [1]. The Walker results continue a theme from this research group (see here) whereby some important data is being generated on how to treat some fairly prevalent bowel issues that seem to accompany quite a few diagnoses of autism (see here).

Much like other research from this authorship group, the research material examined was biopsy tissue - "ascending colon biopsy tissues" - provided by 35 children diagnosed with an autism spectrum disorder "and chronic constipation on a background of enterocolitis." I know some people don't like the word 'enterocolitis' in the context of autism (see here) but prejudices aside, there is nothing in the current research literature to suggest that a diagnosis of autism is somehow protective against the development of inflammatory bowel disease and/or its symptoms. Nothing.

Anyhow, 20 of those 35 children were categorised as 'slow responders' on the basis of showing "recurrent right-sided fecal loading requiring regular colon cleanouts during treatment for enterocolitis" and 15 were defined as 'fast responders' as a function of experiencing "a sustained state of GI [gastrointestinal] symptomatic remission while on maintenance anti-inflammatory therapy." In effect the group was divided up into those whose bowel symptoms got better (n=15) and those whose bowel symptoms did not (even after multiple attempts) (n=20). Researchers analysed those biopsy samples with the expression of genes in mind as per other research occasions [2].

Results: "Significant differences were found between the two clusters with fast responder-predominant cluster showing an upregulation of transcripts involved in the activation of immune and inflammatory response and the slow responder-predominant cluster showing significant over-representation of pathways impacting colonic motility (e.g. genes involved in tryptophan and serotonin degradation and mitochondrial dysfunction)." Apologies for the long quote taken from the Walker paper, but they said it better than I ever could. The translation: gene expression data was different between the fast and slow responder groups.

Obviously more research is needed in this area with larger participant groups and perhaps using samples from other non-autism groups who present (or don't) with various types of bowel issues, whether sensitive to treatment or not. The cluster of genes that were used in the authors' modelling did all right when it came to talk of possible 'biomakers' - "The sensitivity (sensitivity = 0.88), specificity (specificity = 0.89), and kappa (kappa = 0.77) statistics all reflect a good strength of agreement between prediction and actual assignments" - but still need more work before any big claims are made.

There are a couple of other things to mention from the Walker results. So, results suggested that: "predominantly chronic constipation in fast responders is not only related to the inflammatory status of the right colon but is likely a direct consequence of this colonic inflammation." Inflammation perhaps equalling constipation? Interesting. And it not only offers lots more avenues for further study but also some important treatment options.

Next, the amino acid tryptophan was singled out as being potentially "especially significant." I've always been interested in the aromatic amino acids in relation to some autism (see here). Tryptophan is a particularly important aromatic amino acid because it's eventually metabolised into a whole slew of important compounds from serotonin (5-HT) to melatonin and beyond, with some interesting connections to autism (see here). Walker and colleagues mention how: "In the slow responder cluster of patients, there was a significant upregulation of transcripts in each of the metabolic degradation pathways for tryptophan, serotonin, and melatonin, suggesting that TRP [tryptophan] insufficiency (and therefore 5-HT insufficiency) may be an important factor in the sustained hypomotility seen in this patient cohort." There's some much more study that one could do in this area. Particularly when 'gut hypomotility' is a potential issue for quite a few people on the autism spectrum (see here).

There are other things to consider from the Walker paper - "A third relevant theme apparent from the slow response gene expression profile involves a number of pathways that converge in the mitochondria and impact mitochondrial function" - but I'll leave that for now (see here). Suffice to say that there is enough evidence emerging in the peer-reviewed domain to say that (a) pathological bowel problems are more than present alongside a diagnosis of autism, (b) said bowel issues also overlap with functional GI symptoms such as constipation in particular, (c) there are physiological reasons for such bowel issues outside of any psychobabble explanations, and (d) lots more research is required in this area without fear or favour pertinent to improving quality of life...

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[1] Walker SJ. et al. A molecular biomarker for prediction of clinical outcome in children with ASD, constipation, and intestinal inflammation. Sci Rep. 2019 Apr 12;9(1):5987.

[2] Walker SJ. et al. A Putative Blood-Based Biomarker for Autism Spectrum Disorder-Associated Ileocolitis. Sci Rep. 2016 Oct 21;6:35820.

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Carnitine supplementation and autism: "side-effects and behavioral outcomes"

'Favourable outcomes' is a term mentioned in amongst the various findings reported by Robin Goin-Kochel and colleagues [1] following their examination of "dose compliance, attrition, and potential side effects of short-term, high-dose carnitine supplementation" in a small group of boys diagnosed with an autism spectrum disorder (ASD). Understanding that the Goin-Kochel study was primarily directed at looking at safety, on the basis of 'high-dose' carnitine supplementation, it appears that an elevation of plasma carnitine and related metabolites was not the only effect noted in their small cohort (N=10).

Tracking back slightly, carnitine is an important compound. Not quite an amino acid, carnitine plays an important role in energy production; as per use of the word 'mitochondria' and it's transporting duties of long-chain fatty acids to the cell powerhouse for energy conversion. You probably won't be surprised to hear that carnitine has a *connection* to some autism (see here and see here). Indeed, Goin-Kochel et al mention the findings reported by Patrician Celestino-Soper and colleagues [2] and their identification of a genetic issue that impacts on 'carnitine biosynthesis' in some people diagnosed with ASD. At least one of the authors on the Goin-Kochel paper has some pretty important knowledge about that finding of trimethyllysine hydroxylase epsilon (TMLHE) gene issues in the context of autism...

Alongside looking for reports of any side-effects from the use of carnitine - "oral suspension or tablets of levocarnitine in 3 divided doses, starting at 200 mg/kg/day and increasing to 400 mg/kg/day, with a maximum daily dose of 6 g" - various behavioural schedules were included in the study protocol. Some were objective measures of autism symptomatology; others were parent-report measures. The use of the Clinical Global Impression Scale (CGIS) also provided a helpful 'clinicians' overview' of before and after supplementation in this open-trial.

Results: a few side-effects coinciding with carnitine use were reported. These included: "heavy odor (4 parents), diarrhea (4 parents), and sporadic vomiting (1 parent)." Such reported side-effects meant that three children remained at the lower dose of carnitine over the experimental period (8 weeks).

Alongside, a few other 'favourable outcomes' were also reported: "calmer behavior (2 parents), more energy (2 parents), increased prosocial behaviors (4 parents), greater awareness (2 parents), better eye contact (2 parents), and improved language skills (2 parents)." These parental reports were accompanied by some 'changes' noted on the various schedules included in the study protocol, including those CGIS ratings. The authors used the study results produced by Geier and colleagues [3] as their comparator; highlighting how both studies had picked up "improvements in overall ASD symptoms... and some language ratings." Importantly too, Goin-Kochel et al talk about how none of their cohort were rated as "worse at post treatment."

Where next? More research please. Bigger participant numbers, more methodologically sound study designs and perhaps also, investigation of the potential pros-and-cons of carnitine supplementation over a longer period of time. By all means keep an eye on those side-effects and perhaps look to the biochemistry as to why such side-effects might appear; indeed look to the biochemistry for potential best-responders to this type of intervention too ("One child had documented TMLHE deficiency and 3 had low carnitine levels" in the Goin-Kochel cohort). But more study is definitely indicated...

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[1] Goin-Kochel RP. et al. Side Effects and Behavioral Outcomes Following High-Dose Carnitine Supplementation Among Young Males With Autism Spectrum Disorder: A Pilot Study. Global Pediatric Health. 2019; 6: 1-8.

[2] Celestino-Soper PB. et al. A common X-linked inborn error of carnitine biosynthesis may be a risk factor for nondysmorphic autism. Proc Natl Acad Sci U S A. 2012 May 22;109(21):7974-81.

[3] Geier DA. et al. A prospective double-blind, randomized clinical trial of levocarnitine to treat autism spectrum disorders. Med Sci Monit. 2011 Jun;17(6):PI15-23.

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An easily testable blood metabolic profile associated with ASD diagnosis?

"The present study supports early recognition of a distinctive metabolic profile in DBS [dried blood spots] whose distinguishing features suggest a reduced flux through the mitochondrial fatty acid β-oxidation pathway and provides insight into concealed molecular mechanisms determining ASD [autism spectrum disorder]."

So said the findings reported by Rita Barone and colleagues [1] including some notable names on the authorship list with some previous interest in acyl-carnitines and autism (see here), one important source of discussions in the Barone study.

Some basics from the Barone study: "A targeted panel of 45 ASD analytes including acyl-carnitines and amino acids extracted from DBS was examined in 83 children with ASD... and 79 matched, neurotypical (NT) control children." Autism was confirmed as autism in the 83 autistic children, also including the application of some important exclusionary criteria such as a "positive history for mitochondrial disease or known medical conditions including autoimmune disease and inflammatory bowel diseases (IBD)/celiac disease." I was also happy to see that researchers screened for 'possible autism' in their control participants too: "The Social Communication Questionnaire was used to screen and exclude autism in TD [typically developing] children."

Although the Barone study was a study predominantly analysing dried blood spot samples (a sample medium that has always been slightly under-utilised in research circles), researchers did also look at urine and blood samples obtained during the course of their study. They reported some interesting observations as a consequence of analysis; notably that: "Twenty-five out of 40 studied [autistic] subjects (62.5%) had significantly decreased blood Vitamin D3 levels with normal Ca/P ratio." Decreased vitamin D levels are no stranger to autism (see here and see here).

Insofar as the 'ASD analytes' results, we are told that 8 acyl-carnitines were significantly increased in the autism group compared to the control (not autism) group. I'm not going to bore you with the specific details but suffice to say the list of compounds was pretty robust and "confirm the same, unique pattern of acyl-carnitine profile" as noted in other studies with other groups. Researchers also mention how one particular amino acid - citrulline - was also significantly increased in the autism group compared to controls. They talk about how: "Blood citrulline level is considered a biomarker of gastrointestinal mucosal surface and enterocyte integrity" among other things and could have some implications for that and other 'effects'.

And then something else: "The present study confirms that patients with ASD may show a distinct metabolic profile, demonstrating that this can be used to identify a subset of ASD patients with respect to TD at younger ages." I'm always a little bit wary of studies talking about biomarkers and autism (see here for another example) but the important use of the word 'subset' denoting how autism is a label covering significant heterogeneity makes me feel a little easier about such sentiments (see here and see here) albeit with much more study being required.

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[1] Barone R. et al. A Subset of Patients With Autism Spectrum Disorders Show a Distinctive Metabolic Profile by Dried Blood Spot Analyses. Front Psychiatry. 2018 Dec 7;9:636.

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'Comprehensive metabolomics' and ME/CFS: lipid and energy production turn up again

The findings reported by Dorottya Nagy-Szakal and colleagues [1] describing the results of "biomarker discovery and topological analysis of plasma metabolomic, fecal bacterial metagenomic, and clinical data from 50 ME/CFS [myalgic encephalomyelitis/chronic fatigue syndrome] patients and 50 healthy controls" provide the rather long blogging fodder today.

Just in case that opening quote sounds like gibberish, this was a study that in effect examined two quite prominent biological 'systems' alongside looking at symptom profiles of participants diagnosed with ME/CFS compared with controls. Metabolomics is a discipline that is no stranger to this blog, and is focused on the analysis of small molecule metabolities in a range of biological fluids (see here). The interface between the technology used to separate out and analyse said metabolites and the statistical analysis of the huge amounts of data generated as a result, are what make metabolomics the science that it is. 'Fecal bacterial metagenomics' also known as microbiomics (see here) refers to the science of cataloguing what bacterial species are present in poo(p) samples. Yes, bacteria have their own genomes too, and stool samples can therefore be a rather informative medium.

It's important to realise that this isn't the first time that metabolomics has been spoken in the same breath as CFS/ME (see here and see here for examples); something alluded to in the Nagy-Szakal paper. Indeed, this most recent paper adds to the authors other work in this area [2] (see here for my take) where the focus was on immune-related parameters and their *association* with CFS/ME in the context of the gut and its bacterial inhabitants. And once again, there are some eminent research names included on the authorship list as last time...

So, fifty participants diagnosed with CFS/ME were compared with 50 asymptomatic (I hate the words 'healthy control') participants, and their blood (plasma) and stool were analysed. Mass spectrometry played an important role in their metabolomic work, as over 550 compounds were initially separated out from the samples provided and identified.

Results: "Among the top plasma biomarkers differentiating ME/CFS patients from controls were decreased levels of betaine, complex lipids (lysophosphatidylcholine [LPC], phosphatidylcholine [PC]) and sphingomyelin (SM), and increased levels of triglycerides (TG), α-N-phenylacetyl-glutamine, ε-caprolactam and urobilin." I'm not going to go through all of those compounds individually as to their possible relevance but there are some important classes of compound being mentioned (i.e. lipids and triglycerides).

Authors also mention another group of compounds as also potentially being important: ceramides. You may have heard the word 'ceramide' before if you are/were a user of certain brands of shampoo in recent times (see here). Outside of any hair care role, ceramide "is a waxy lipid implicated in suppression of electron transport, insulin and leptin resistance and apoptosis." Among the many roles they play 'in' the body, there is some research literature to suggest that ceramides "may play a role in gut barrier dysfunction and increased gut permeability." Interesting (see here). And going back to the Nagy-Szakal results we are told that "patients with ME/CFS and IBS [irritable bowel syndrome] have increased plasma levels of ceramide." Even more interesting.

Having mentioned the gut and gut issues in the form of IBS, it's also important to note that the authors made allowances for the presence of such gut dysfunction in their participant groups. And yes, one needs to remember that it was "based on self-reported diagnosis of IBS on the medical history form". As probably expected, the introduction of IBS (self-reported) did seem to affect the metagenomic (microbiomic) data obtained (something authors talked about in their last paper). More than that: "Chemical enrichment analysis of plasma metabolites revealed that metabolomic profiles of ME/CFS patients with IBS were distinguished from controls by levels of TG, ceramides, phosphatidylethanolmines (PE) and metabolites in the carnitine-choline pathway." Indeed also, take away the IBS bit from the ME/CFS findings and: "ME/CFS patients without IBS co-morbidity showed disturbances in PCs and carnitine-choline pathways, similar to the disturbances found in the overall ME/CFS cohort." Again, interesting.

Authors conclude that their results draw attention to a few areas already pertinent to CFS/ME, in particular, "lipid and energy metabolism." The word 'mitochondria' figures a few times in their results write-up and specifically how: "compounds in the choline-carnitine pathway were decreased in ME/CFS patients regardless of their IBS status." I've written about quite a bit of research on mitochondria and CFS/ME (see here and see here for examples) and how even if there aren't genetic reasons for mitochondrial issues (see here), this does not mean that there may not be more other issues with this system (see here).

We really need much more research in the area of metabolomics and ME/CFS. And patients really need it now, not some time later in the future...

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[1] Nagy-Szakal D. et al. Insights into myalgic encephalomyelitis/chronic fatigue syndrome phenotypes through comprehensive metabolomics. Sci Rep. 2018 Jul 3;8(1):10056.

[2] Nagy-Szakal D. et al. Fecal metagenomic profiles in subgroups of patients with myalgic encephalomyelitis/chronic fatigue syndrome. Microbiome. 2017; 5: 44.

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KPAX002 for Chronic Fatigue Syndrome part 2: controlled study says no

KPAX002 mentioned in the title of this post refers to "a mitochondrial modulator technology platform" according to the manufacturer that includes a low dose of methylphenidate combined with various nutrients designed to impact on mitochondrial function. Within the context of chronic fatigue syndrome (CFS) also known as myalgic encephalomyelitis (ME) (but not necessarily accurately so!), there is some preliminary research history suggesting that KPAX002 might be something to look at for intervening in some of the disabling characteristics of CFS/ME (see here). This, on the basis that mitochondria in particular, might be something quite important to at least some cases (see here and see here).

The fly in the scientific ointment?

Well the results of the "phase 2 randomized, double-blinded, placebo-controlled trial" on KPAX002 published by Jose Montoya and colleagues [1] that, from an intention-to-treat point of view, reported no significant statistical difference in self-reported group scores of fatigue and other measures between active treatment and a placebo. In keeping with the phase 2 label attached to the trial - looking at both initial clinical results and also any side- or adverse effects - authors reported no statistically significant difference in the frequency of reported adverse effects between KPAX002 and a placebo over the 12 weeks of study. First, do no harm and all that.

The Montoya paper is open-access so readers can see for themselves how things were done and the details of the results. I however, want to highlight a few points that I thought were important:

First, the authors acknowledge the "unexpectedly positive results" observed the last time around [2] that led to this more rigorous trial. Personally, I don't think there was anything too unexpected about those pilot study results, given the methodological issues typically associated with a pilot study. Y'know, a small un-blinded participant group taking part in a trial using a preparation that they probably will have been told *might* affect various symptoms they experience or themselves possibly 'exposed' to other anecdotal reports of good effects. That and no control group, no placebo included and importantly, no objective measure of fatigue (a real issue when it comes to quite a bit ME/CFS research) and well, I'd be surprised if something significant didn't come up during the initial findings. And just in case you think I'm being all 'high-and-mighty' about this, I've published using the same type of pilot study methodology before, including some of the same inherent issues (see here).

Second, I'm a little bit disappointed that the authors weren't more forthright in how the results weren't statistically significant on any and all measures included for study. I say this on the basis of both the commercial take on the results (see here) and also sentences like: "The two groups demonstrating the most robust response to KPAX002 were subjects with more severe ME/CFS symptoms at baseline (P=0.086) and subjects suffering from both fatigue and pain (P=0.057)." Both those p-values (p being a measure of statistical significance) are above the [currently] recognised threshold for p equal to or less than 0.05, yet are listed as a 'robust response'. Even more, throughout the paper I note the words 'trend in favor of' being used, which some people might translate as being 'well, they were nearly statistically significant results'. I say this also bearing in mind that the final participant numbers - KPAX002 use = 48 and placebo = 57 - are not exactly facets of what one would call an under-powered study. I'm probably being a nit-picker here but like it or not, the [current] rules of science are the [current] rules of science.

Finally, once again, I note that under the heading 'Disclosure of conflict of interest', the word 'none' appears as per the last research occasion [2]. Personally, and with no malice intended, I would have listed the detail that at least one of the authors is an employee of the manufacturer of KPXA002 given the affiliation details and email address for further correspondence provided on the paper. Again, it's a small detail but one that should nevertheless be acknowledged. I would have also like to have seen a little more on who funded the trial too and especially who funded the provision of the KPAX002 supplement for trial purposes. I reiterate that there is no malice is intended in saying that, but readers require such details.

I don't want to come down too hard on these results because it's obvious that quite a bit of work has gone into their production. I'm also not closing the door on the idea that future research with a more targeted group with ME/CFS might not produce something a little more statistically significant with regards to KPAX002. But for now, the answer must be that controlled study of the formulation did not meet clinical endpoints in a statistical sense, and hence KPAX002 cannot be said to be superior to placebo for CFS/ME. With all the setbacks that the label(s) ME/CFS has had to endure down the years with regards to the 'psychobabble' explanations (see here) and other 'eureka' moments (see here), the Montoya findings are bad news for patients yet again. But, they also should represent a further call to re-double research efforts; particularly when it comes to the biology of the condition(s) and onward the acceleration of research for interventions for this quality of life draining condition (see here).

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[1] Montoya JG. et al. KPAX002 as a treatment for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): a prospective, randomized trial. Int J Clin Exp Med 2018;11(3):2890-2900

[2] Kaiser JD. A prospective, proof-of-concept investigation of KPAX002 in chronic fatigue syndrome. Int J Clin Exp Med. 2015 Jul 15;8(7):11064-74.

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