"serum levels of certain endocannabinoids are substantially decreased in people with ASD"

The quote titling this post - "serum levels of certain endocannabinoids are substantially decreased in people with ASD [autism spectrum disorder]" - comes from the paper published by Adi Aran and colleagues [1]. It adds to previous study on this topic (see here) and continues a research theme from members of this authorship team where the word 'cannabis' is being discussed - in the peer-reviewed science domain - in the context of [some] autism (see here).

Distinct from the last time authors' research appeared on this blog talking about the feasibility of "Cannabidiol-Rich Cannabis" 'for autism' [2], the name of the research game this time around was to assess "the circulating levels of several endocannabinoids and delineate the correlations between their levels and disease characteristics in a large group of children with ASD and their matched controls with typical development." Researchers mention how previous studies in this area "were not designed to comprehensively characterize the involvement of the ECS [endocannabinoid system] in the pathogenesis of ASD" in quite a sweeping blow to some of the other research in this area.

So, endocannabinoids are part of a system that is involved in various important biological processes [3]. As the name suggests there's an overlap between 'endogenous cannabinoids' and some of the chemical components seen in cannabis that provides as good an answer as any as to why cannabis use/misuse is the continuing issue that it is in a population sense. Authors talk about their study focusing on various endocannabinoids: AEA (anandamide), 2-AG (2-arachidonoil-glycerol), AA (arachidonic acid), PEA (N-palmitoylethanolamine), and OEA (N-oleoylethanolamine). They report how said compounds in serum samples were "analyzed by liquid chromatography/tandem mass spectrometry in 93 children with ASD... and 93 age- and gender-matched neurotypical children." Please don't however get me started on the nonsense that is the word 'neurotypical' (see here). Various other behavioural, psychometric and demographic data were also collected and thrown into the statistical mix.

Results: "Serum levels of the main endocannabinoid AEA and its structurally related compounds OEA and PEA were lower in children with ASD versus age-, gender-, and BMI [body mass index]-matched control group of typically developed children." Nothing particularly new there, as the lower levels of anandamide for example, mimic those reported by Karhson and colleagues [4]. Researchers also mentioned how their findings *might* also have some other potential: "circulating AEA, OEA, and PEA might be used to identify a biologically homogeneous subgroup of ASD, predict response to treatments and adverse reactions to medications, and assist in the development of novel drugs that target specific core symptoms of ASD." Interestingly, some of these 'options' have already been explored [5] in humans and also some animal models [6] with autism in mind.

As to the biochemical *links* between the Aran findings and indeed, the ECS more generally with autism, well, there's still a way to go to decipher them all yet. There are clues emerging [7]; clues that intersect with other important autism-relevant concepts like inflammation among other things. I note also the authors mention how their findings "support the rationale in the ongoing and emerging clinical trials of CBD [cannabidiol] in ASD" (see here) and some results to come.

I'm well and truly [cautiously] interested...

----------

[1] Aran A. et al. Lower circulating endocannabinoid levels in children with autism spectrum disorder. Molecular Autism. 2019; 10:2.

[2] Aran A. et al. Brief Report: Cannabidiol-Rich Cannabis in Children with Autism Spectrum Disorder and Severe Behavioral Problems-A Retrospective Feasibility Study. J Autism Dev Disord. 2018 Oct 31.

[3] Lu HC. & Mackie K. An Introduction to the Endogenous Cannabinoid System. Biol Psychiatry. 2015;79(7):516-25.

[4] Karhson DS. et al. Plasma anandamide concentrations are lower in children with autism spectrum disorder. Mol Autism. 2018 Mar 12;9:18.

[5] Antonucci N. et al. Beneficial Effects of Palmitoylethanolamide on Expressive Language, Cognition, and Behaviors in Autism: A Report of Two Cases. Case Rep Psychiatry. 2015;2015:325061.

[6] Servadio M. et al. Targeting anandamide metabolism rescues core and associated autistic-like symptoms in rats prenatally exposed to valproic acid. Transl Psychiatry. 2016 Sep 27;6(9):e902.

[7] Brigida AL. et al. Endocannabinod Signal Dysregulation in Autism Spectrum Disorders: A Correlation Link between Inflammatory State and Neuro-Immune Alterations. Int J Mol Sci. 2017;18(7):1425. Published 2017 Jul 3.

----------

Autism and the measurement of urinary amino acids

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

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

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

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

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

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

----------

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

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

----------

"Starting this week, the first blood test for autism will be available to the public"

I have to say that of all the news outlets that I peruse now and again, the resource known as Disability Scoop is typically one of the best. They just always seem to be 'on the ball'. The headline titling this post - "Starting this week, the first blood test for autism will be available to the public" - comes from that resource and well, 'whoa' is a word that springs to mind.

The report details that a company called NeuroPointDX is "launching its NPDX AA test, a blood plasma test that screens for certain metabolic markers that the company has linked to autism spectrum disorder." Said test is based on some peer-reviewed science by Alan Smith [1] which concluded that the: "Identification and utilization of metabotypes of ASD [autism spectrum disorder] can lead to actionable metabolic tests that support early diagnosis and stratification for targeted therapeutic interventions." And before you ask, yes, I have covered the Smith paper before on this blog (see here).

Specific details of what is included in the NPDX AA test are, at the time of writing, not seemingly readily available. The 'AA' mention in the test name implies amino acids are going to be central to the analysis. Indeed, the Smith paper [1] talked about a few specific amino acids as potentially being important: "The combination of glutamine, glycine, and ornithine AADMs [Amino Acid Dysregulation Metabotypes] identified a dysregulation in AA/BCAA [branch chain amino acids] metabolism that is present in 16.7% of the CAMP [Children’s Autism Metabolome Project] ASD subjects and is detectable with a specificity of 96.3% and a PPV [positive predictive value] of 93.5%." What this translates into is that for at least one part of the very heterogeneous autism spectrum (maybe one or more of the autisms?), this test might be able to identify some metabolic issues that could be considered both diagnostic (for that particular 'type of autism') and also therapeutic, insofar as specific interventions aimed at specific amino acid 'issues' when identified (see here for one possible example).

Of course we've kinda been here before with the talk about a biological test for autism (see here for example) and history teaches us to be quite cautious when it comes to such discussions. We'll just have to see how well the NPDX AA test does 'in the field' before any further claims are made and even bigger 'shifts' in our knowledge of autism reported and accepted. But hey, at least give it a chance...

To close, your customary 'chat' from V to remember the date today...

----------

[1] Smith AM. et al. Amino acid dysregulation metabotypes: potential biomarkers for diagnosis and individualized treatment for subtypes of autism spectrum disorder. Biological Psychiatry. 2018. Sept 6

----------

The Children’s Autism Metabolome Project (CAMP) reports: "Amino acid dysregulation metabotypes"

The Children’s Autism Metabolome Project (CAMP), mentioned in the title of this post, is an initiative that aims to develop "a diagnostic blood test for autism." It's a project that grabbed my attention for a few reasons; not least the reliance on the science of metabolomics and the use of some pretty amazing technology headed under the term 'mass spectrometry' (see here for another example) to try and accomplish their goal.

The recent results published by Alan Smith and colleagues [1] provide some of the first results to come from the CAMP, and the observation that: "Identification and utilization of metabotypes of ASD [autism spectrum disorder] can lead to actionable metabolic tests that support early diagnosis and stratification for targeted therapeutic interventions." Just in case you were wondering: "A metabotype is a subpopulation defined by a common metabolic signature that can be differentiated from other members of the study population." Inevitable lay media headlines have also followed on from this work (see here).

The nuts and bolts? Well: "Dysregulation of AA metabolism was identified by comparing plasma metabolites from 516 children with ASD with those from 164 age-matched typically-developing (TYP) children recruited into CAMP." AA refers to amino acids, the biological building blocks of proteins, and how, yet again (see here and see here) these compounds might be quite important to at least some autism. Researchers were able to analyse blood (plasma) samples from the participants, pertinent to detecting various amino acids and looking at how levels might differ as a function of a diagnosis of autism. Interestingly and importantly, the words "Training and Test Sets" are also used in the Smith paper, denoting how: "A training set was used to identify metabotypes associated with ASD and a test set was used to evaluate the reproducibility of the metabotypes." Similar methodological processes have been noted in other autism metabolomic studies (see here). I'm not going to bore you with the technological details of the "Triple Quadrupole LC-MS/MS Method" used (I'm more inclined to q-ToF mass spec myself) but suffice to say that such technology did yield some pretty accurate and important results, and it wasn't all just about autism vs. not-autism either.

"A simple analysis of the mean concentrations of free plasma amines did not reveal meaningful differences between the ASD and TYP populations of children." This is an important point. It suggests that within this cohort, there was no significant difference in the biological profiles following a straight 'autism vs not-autism' analysis. Something perhaps not entirely unexpected given the significant heterogeneity under the behaviourally-defined label called autism. But... "scatterplots of amine levels indicated that there were subsets of children with ASD with amine levels at the extreme upper or lower end of the abundance distribution." Researchers then began zooming in on different sub-groups of their autistic cohort as part of their "Amino Acid Dysregulation Metabotype (AADM)" description. Such analysis revealed a few AADMs based on the ratios between various amino acids. Further: "Taken together, all AADMs identified an altered metabolic phenotype of imbalanced BCAA [branched chain amino acid] metabolism in 16.7% of CAMP ASD subjects with a specificity of 96.3% and PPV [positive predictive value] of 93.5%." This *could* be interpreted as suggesting that about 15% of kids with autism *could* be correctly identified via their amino acid profile.

Caveats? Well yes, a few. This was work, for example, based on a single blood sample from each participant, in effect, providing a snapshot of each person at a particular point in time. There are lots and lots of different variables that will affect our metabolome including health/illness, diet, exercise, any medicines taken, comorbidity, et al. It's not beyond the realms of possibility that any or all of those factors could have influenced the results both in the short- or longer-term. Indeed, I'd like to see a lot more research on the consistency of individual sample results across different time frames before any big claims about a diagnostic test for autism are made. Also, the term 'biomaker for autism': I again get the impression that there needs to be lots more 'cross-linking' discussion between groups committed to this research agenda (see here and see here for examples).

But I don't want to take anything away from this work and (hopefully) future publications to come the CAMP. And I do also want to mention a couple of other interesting snippets of information garnered from the current study. So, in one of the write-ups of the study, one of the authors who is not stranger to the concept of 'biomarkers for autism' (see here) discusses: "Amaral points to phenylketonuria (PKU) as a possible template. PKU is a rare disease in which the amino acid phenylalanine builds up, causing brain damage. However, relatively small dietary adjustments can make a big difference." PKU as a template for autism? I think I've heard that somewhere before (see here). And that's also to acknowledge that PKU and autism can very much exist together (see here) in the context that various inborn errors of metabolism seem to be able to produce autistic signs and symptoms (see here). And some of them are very treatable...

Also alongside, I must quickly mention about those branched-chain amino acids (BCAAs) highlighted by Smith et al. How, in the context of other previous important research talking for example, about a 'new form of autism found' (see here), there are lots and lots of research (and clinical) possibilities to come from the analysis of these types of amino acids in the context of autism (see here). And yes, this includes intervention...

----------

[1] Smith AM. et al. Amino acid dysregulation metabotypes: potential biomarkers for diagnosis and individualized treatment for subtypes of autism spectrum disorder. Biological Psychiatry. 2018. Sept 6.

----------

On postpartum paracetamol metabolites and risk of offspring ADHD: details matter

The findings reported by Yuelong Ji and colleagues [1] provide the blogging fodder today, as once again the topic of acetaminophen (a.k.a paracetamol) use during pregnancy and offspring risk of attention deficit hyperactivity disorder (ADHD) is in the spotlight. I say 'once again' because this topic has cropped up time and time again on this blog (see here for example).

On this research occasion, the starting point was the Boston Birth Cohort (a cohort that has been mentioned before) and specifically, an analysis of "maternal plasma acetaminophen metabolites levels measured within a few days after delivery and ADHD diagnosis in the offspring" as a measure of paracetamol exposure. The metabolites in question were "unchanged acetaminophen, acetaminophen glucuronide, and 3-(N-Acetyl-l-cystein-S-yl) acetaminophen." All were measured in blood plasma using one of the gold-standard chemical analytical techniques: mass spectrometry. A diagnosis of ADHD was extracted from medical records on the basis of ICD-9 and/or ICD-10 definitions.

Results: taking into account other potential confounding variables, authors reported finding "a significant positive association between maternal blood acetaminophen metabolite levels measured within 1–3 days postpartum and ADHD diagnosis in offspring." It's perhaps however important to understand how such a finding was arrived at, bearing in mind that ADHD was not the only diagnostic fruit examined in the Ji study. So: "The main exposures analyzed in this study were maternal acetaminophen metabolite levels, which were inverse normal transformed to approximate the normal distribution." What this means is that rather than reporting the specific levels of each paracetamol metabolites across different diagnoses and a 'neurotypical group' ("Children without any diagnosis of ASD [autism spectrum disorder], ADHD, developmental delays, or intellectual disabilities were classified as neurotypical (NT)"), authors chose to covert the raw values into groupings. Groupings were based around "no detection, below median, above median of detected values." I'm not altogether sure that this is the best way to report results; certainly I would have liked to have seen the raw values for each metabolite according to group as a comparator; but that's just my research preference. Researchers mention that they did not see any association between paracetamol metabolites and any other diagnostic label, and also stressed how the association remained when other potentially confounding variables were taken into account. And when it comes to ADHD, there are a few (see here for example).

How much weight can we give these results? Well, this is probably the first time that someone has looked at actual paracetamol metabolites in mums rather than just relying on maternal or doctor records of paracetamol usage during pregnancy, and that's a good thing. Authors also mention that their study was "further strengthened by the diagnosis of ADHD by both general pediatricians and developmental specialists." Add in the prospective design of the study, and you have some potentially important evidence for a *relationship* between paracetamol use and subsequent offspring development.

But... "this study only included a one-time measurement of maternal acetaminophen metabolite levels within 1–3 days postpartum." The authors rightly acknowledge that paracetamol is pretty quickly metabolised in the body so, at best, their study really only looked at recent paracetamol use immediately during or after birth. Maybe further study of archived samples during pregnancy (at multiple points during pregnancy) would provide some further information? And comments from the authors like "women with detectable levels of acetaminophen biomarkers are likely to be more regular users" really don't have any place in a scientific paper without supporting evidence. Particularly when pain relief is going to be pretty important when it comes to the process of childbirth.

Also: "our metabolite measurement method did not include acetaminophen sulfate, which accounts for 30–44% of the total metabolites of acetaminophen under the normal dosage." I don't want to go into the nitty-gritty of paracetamol metabolism, but just going back to the research looking at sulphation (sulfation) in the context of autism (see here), it would have been useful to examine both paracetamol sulphate and paracetamol glucuronide at the same time. Certainly it would have provided a more complete picture of typical paracetamol metabolism.

Don't get me wrong, I am interested in the Ji findings and their addition to the literature on paracetamol use and offspring outcomes. This still remains an important area of investigation. I'm not however completely convinced that the evidence presented by Ji and colleagues counts as particularly strong evidence despite the important measurement of paracetamol metabolites in this context...

----------

[1] Ji Y. et al. Maternal Biomarkers of Acetaminophen Use and Offspring Attention Deficit Hyperactivity Disorder. Brain Sci. 2018 Jul 3;8(7). pii: E127.

----------

'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...

----------

[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.

----------

"A strict and lifelong gluten-free diet can help recover vitamin D level without any supplementation"

The quote titling this post - "A strict and lifelong gluten-free diet can help recover vitamin D level without any supplementation" - comes from the results published by Fabiana Zingone & Carolina Ciacci [1].

These authors inspected the peer-reviewed literature looking at vitamin D levels in relation to the archetypal 'diet can affect health' autoimmune condition that is coeliac (celiac) disease. They looked at both something called calcifediol, otherwise known as 25-hydroxyvitamin D (25(OH)D), and also something called calcitriol, also known as 1,25-dihydroxyvitamin D3. The difference between the two 'forms' of vitamin D is that one represents the 'pre-hormone' version  - (25(OH)D) - that is typically quantified to give a 'where you're at' measurement of biological vitamin D levels, and the other - 1,25-dihydroxyvitamin D3 - is the 'active' metabolite. Through the wonders of something called mass spectrometry, these and other 'forms' of vitamin D are able to be quite accurately measured [2] in various biological fluids.

Zingone & Ciacci reported that "most of the studies on vitamin D in adult CeD [coeliac disease] report a 25 (OH) vitamin D deficiency at diagnosis that disappears when the patient goes on a gluten-free diet, independently of any supplementation." This finding really intrigued me. It got me thinking of a few things; notably about mechanisms and biological relationships and indeed, how applicable such a finding could be to the general 'not coeliac' population or those 'around the diagnostic edges' of CeD.

OK, it should be noted that part-and-parcel of CeD is an issue with malabsorption, where 'the body does not fully absorb nutrients' (see here). The inflammatory processes at work in CeD do some pretty awful things to the structure and functioning of the mucosal lining of the bowel which aren't really conducive to optimal absorption of nutrients from food. The implementation of a gluten-free diet does help matters; and so logic dictates that absorption of something like vitamin D from food sources will be improved when a gluten-free diet is implemented. I've also blogged about how this process *may* also be part of the effect noted in relation to [some] autism and the use of similar dietary intervention strategies (see here). Alongside, I'll mention that, done right, a gluten-free diet is actually not the most unhealthiest diet in the world either.

Having already sort-of mentioned something like non-coeliac gluten/wheat sensitivity in the contest of the 'diagnostic edges' of CeD, I do wonder if there could be a further plan of research there too. Y'know to look at questions like whether such not-quite-coeliac-disease conditions also (a) manifest as having low levels of vitamin D when not diet treated and (b) whether the use of a gluten-free or other diet (see here) might similarly positively effect vitamin D levels in those circumstances?

There's also another potentially important explanation to consider which was tweeted by Dr Emily Deans (she of the fabulous Evolutionary Psychiatry blogs): could vitamin D deficiency be a marker of something more general? To quote from Dr Deans' tweet (shown pictured above): "Because it’s [vitamin D] an inverse acute phase reactant and goes down with illness and up with health." There is some sound logic behind such an observation insofar as vitamin D insufficiency/deficiency being associated with all manner of physical and behavioural/psychiatric diagnoses/conditions/states (see here and see here for examples). Correction of vitamin D deficiency is all well and good when it comes to correcting biological measures, but outside of treating something like rickets, the evidence for extra-skeletal effects from such supplementation is currently not that great.

Finally, I'll draw your attention back to some work suggesting that vitamin D might itself have some interesting effects on gut barrier function (at least in mice) (see here). One wonders what this could also mean for CeD and the spectrum of gluten-related issues?

-----------

[1] Zingone F. & Ciacci C. The value and significance of 25(OH) and 1,25(OH) vitamin D serum levels in adult coeliac patients: A review of the literature. Dig Liver Dis. 2018 Apr 13. pii: S1590-8658(18)30702-3.

[2] van den Ouweland JM. et al. Vitamin D and metabolites measurement by tandem mass spectrometry. Rev Endocr Metab Disord. 2013 Jun;14(2):159-84.

----------

Baby teeth, metals and autism part 2

Consider this post on the findings reported by Paul Curtin and colleagues [1] which concluded that "altered zinc-copper rhythmicity precedes the emergence of ASD [autism spectrum disorder]" an extension of other previous discussions on this blog (see here). The research starting material was, once again, baby teeth, including teeth derived from an interesting initiative called "the Autism Tooth Fairy Project" based in Texas, USA.

On the last blogging occasion, the paper by Manish Arora and colleagues [2] was central to my discussions, and the use of some really interesting analytical technology to look at levels of various metals in tooth layers: Laser ablation-inductively coupled plasma mass spectrometry. Fast forward a year or so, and authors (including Arora) moved on to bigger and better things, this time focusing specifically on the metals zinc and copper "from the second trimester to approximately 1 year postnatally." Such a feat was achieved because baby teeth, much like the rings of a tree, provide a sort of 'album' of levels of such metals over a child's earliest years. Both these metals and their ratio also have some previous research 'form' when it comes to autism (see here).

"Our participants were recruited from four different studies being undertaken in three countries." Yep, so alongside teeth from the RATSS study initiative (aptly named the 'roots' of autism/ADHD study) forming a discovery dataset, authors also examined teeth gathered as part of other research initiatives including a favourite here in Blighty: the Avon Longitudinal Study of Parents and Children (ALSPAC). These non-RATSS teeth formed a replication set (to try and replicate the data found in the discovery set). Add in some nifty statistical analysis and set lasers to stun or should that be ablate...

Results: authors reported finding "strong evidence for abnormalities in zinc-copper cycles in ASD characterized by shorter duration, lower complexity, and less determinism." A quick translation: well, I'll be honest and tell you that I'm not exactly sure what that actually meant. I think it had something to do with the way the data were analysed, being "analogous to a spectrogram." Suffice to say that levels of copper and zinc were seemingly different in those diagnosed with autism compared with not-autism controls at different tooth time periods. When all this metal data was analysed using some statistics designed to try and 'classify' those with autism from those not-autistic, authors also noted zinc-copper cycles in teeth "allowed for a robust classification of ASD cases and controls." So: "Using optimal threshold criteria, this model was 90% accurate in predicting ASD cases, with 100% sensitivity for ASD diagnosis and 85% specificity to controls." Those stats aren't bad when one considers the importance of sensitivity and specificity to any classifying 'test'.

Although interesting, one has to bear in mind that this research was based on the use of baby teeth and their possible 'retrospective' association with autism. Nobody is advocating pulling out baby teeth as some sort of autism test! The focus on metals however, and specifically "measures of metal rhythmicity" provides some welcome information about how such 'rhythms' *might* show differences in relation to autism. This could reflect things like differing 'exposure' patterns or, more likely, some innate differences in the biological 'processing' of such metals. On that last point, this work potentially fits in with what is emerging in other independent data: metal (various metals) metabolism in the context of some autism seems to be 'unusual' (see here).

----------

[1] Curtin P. et al. Dynamical features in fetal and postnatal zinc-copper metabolic cycles predict the emergence of autism spectrum disorder. Science Advances. 2018. 30 May.

[2] Arora M. et al. Fetal and postnatal metal dysregulation in autism. Nat Commun. 2017 Jun 1;8:15493.

----------

ALSPAC does... prenatal mercury exposure and autism or autistic traits

The ALSPAC - Avon Longitudinal Study of Parents and Children - mentioned in the title of this post is something of quite a regular feature on this blog (see here and see here for examples).

On this particular blogging occasion I'm heading into the findings reported by Jean Golding and colleagues [1] who utilised this fabulous research resource to examine whether "prenatal exposure from total maternal blood Hg [mercury] in the first half of pregnancy is associated with the risk of autism or of extreme levels of autistic traits." They concluded that there were "no adverse effect of prenatal total blood Hg on autism or autistic traits provided the mother ate fish."

OK, mention of the heavy metal mercury in the context of autism and/or autistic traits can be a touchy subject for some. I'm talking about the various 'discussions' that have taken place both in the lay and peer-reviewed science arenas concerned with the exposure patterns relevant to mercury in the context of autism (see here and see here). This, on the basis that mercury exists in several 'forms', and those different forms have different potential exposure routes.

Golding et al relied on some of the gold-standard analytical methods for the analysis of whole blood Hg collected in the most part "at < 18 weeks gestation": "inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS)." Variations on this method - ICP mass spectrometry - have been previously reported on in the context of mercury and autism research (see here and see here). Alongside, they looked at measured levels of mercury in relation to various behavioural and other variable groupings: "(1) direct comparison of 45 pregnancies resulting in children with diagnosed autism from a population of 3840, (2) comparison of high scores on each of the four autistic traits within the population at risk (n~2800), and (3) indirect measures of association of these outcomes with proxies for increased Hg levels such as frequency of fish consumption and exposure to dental amalgam (n > 8000)." They however cautioned that: "Although we accounted for several important confounders which are relevant to Hg levels and autism, the possibility of unmeasured confounding cannot be ruled out." I can think of one potential confounder that was not seemingly included in their list outside of fish consumption and dental amalgams but ho-hum...

Alongside their overall 'no relationship' results, a few other details are noteworthy. First: "all correlations indicated that with increasing levels of [maternal] mercury, the signs of autism [in offspring] were slightly less, but none were statistically significant." Interesting idea - higher maternal levels of mercury during pregnancy 'correlates' with 'less' autistic traits in offspring in childhood - but to reiterate, not statistically significant. Second was that 'provided the mother ate fish' detail attached to the main findings. So: "we have shown a differential relationship between the social cognition trait and prenatal Hg exposure, such that there was a significant difference in apparently protective effects contingent upon whether the mother ate fish." The authors opine as to what it is about fish consumption that might "counteract any possible adverse cognitive and behavioral differences that may be caused by prenatal exposure to Hg" including "the beneficial components of fish such as the omega-3 fatty acids, iodine, and vitamins D and B2." This in the context that omega-3 fatty acids have some research form in relation to autism (see here) as does the sunshine vitamin/hormone that is vitamin D (see here).

One has to be slightly careful with the Golding results given the focus on prenatal exposure, and prenatal exposure at only one early point in pregnancy, as well as also not actually looking at mercury levels in the children themselves. The current results say nothing for example, about any possible direct or acquired role for mercury in relation to autism as per other findings published during the same period [2]. Neither do they offer any additional information on the idea that exposure issues to such heavy metals may be only one part of the story, and that the biological processes involved in removing such heavy metals may be somehow perturbed in relation to some autism (see here).

But... set within the idea that prenatal mercury exposure may be linked to the 'etiology' of at least some autism, the Golding findings represent pretty strong evidence suggestive of no connection.

Music to close, and could I recommend the soundtrack to Sonic 3 while you work?

----------

[1] Golding J. et al. Prenatal mercury exposure and features of autism: a prospective population study. Molecular Autism. 2018; 9: 30.

[2] Qin YY. et al. A comparison of blood metal levels in autism spectrum disorder and unaffected children in Shenzhen of China and factors involved in bioaccumulation of metals. Environ Sci Pollut Res Int. 2018 Apr 22.

----------

Plasma anandamide concentrations are lower in children with autism

The findings reported by Debra Karhson and colleagues [1] piqued my interest for two primary reasons. First, they detail "the first empirical human data to translate preclinical rodent findings to confirm a link between plasma anandamide concentrations in children with ASD [autism spectrum disorder]." Second, authors also report on the use of a gold-standard technique when it came to their analyses: the development and use of "a LC-MS/MS [liquid chromatography-tandem mass spectrometry] method to quantitatively analyze AEA [anandamide] concentrations in small volumes of banked plasma with short sample preparation time and high sample repeatability."

OK, the basis for the Karhson study was the 'increasing interest for ASD' examining the endogenous cannabinoid or endocannabinoid system. This is a system, an internal system, that comprises of quite a few compounds, enzymes and receptors that play "important roles in central nervous system (CNS) development, synaptic plasticity, and the response to endogenous and environmental insults" [2]. Yes, as the name suggests, there is an 'overlap' between some of the workings of the endocannabinoid system (ECS) and components of a certain drug of abuse but that doesn't insinuate anything at the present time.

Anandamide (AEA) is a sort of messenger molecule that is part of the ECS. It shares some chemical characteristics with the active compound found in cannabis, leading quite a few commentators to talk about AEA in terms of being a 'pleasure' or 'bliss' molecule. It does not however, have the 'staying power' of its molecular companion; chemically-speaking being fairly readily degraded in the body. Indeed, of the many biological roles and functions linked to AEA and the ECS more generally, I'd in particular, like to direct your attention to some of the science-so-far literature in relation to pregnancy (see here). And, no, that does NOT mean that smoking marijuana during pregnancy is a good thing...

The authors highlight how the ECS is a research area rising in relation to autism (and associated diagnoses) based, quite extensively, on animal models of autism and all the associated 'issues' that this carries (see here). So: "despite the promise of these preclinical data, no studies to date have investigated AEA concentrations in humans with ASD." They sought to remedy that situation.

Results are reported based on the LC-MS/MS analysis of plasma samples provided by some 59 children with autism and 53 not-autism controls. As per my continued interest in all-things mass spec when specifically applied to autism research, I was encouraged by the use of a "commercially available stable isotope-labeled AEA-d8" being used as an internal standard, and the fact that the lower limit of detection for AEA was in the femtogram range. In short, authors were able to train their system to specifically look for AEA and were able to get down to some really quite low levels of detection.

"Two significant findings were observed: (1) plasma AEA concentrations significantly differentiated ASD cases from controls, such that children with lower AEA concentrations were more likely to have ASD, and (2) AEA concentrations were significantly lower in ASD compared to control children." I don't really need to say much more than that, aside from adding in another quote: "These results, although preliminary, corroborate preclinical evidence that AEA signaling may be impaired in patients with ASD."

The question of what these findings actually mean is still unanswered. It should for example, be noted that the Karhson results in real people were based on the analysis of plasma samples, where previous animal work has tended to be a little more 'invasive' in terms of the tissue types looked at (e.g. in the brain). The authors also mention that further investigations need to give due credit to the idea that autism rarely exists in some sort of diagnostic vacuum (see here), and some of these 'comorbidities' could very well influence the results obtained [3].

Further studies on this topic are very much required.

----------

[1] Karhson DS. et al. Plasma anandamide concentrations are lower in children with autism spectrum disorder. Molecular Autism. 2018; 9: 18.

[2] Lu H-C. & Mackie K. An introduction to the endogenous cannabinoid system. Biological Psychiatry. 2016;79(7):516-525.

[3] Romigi A. et al. Cerebrospinal fluid levels of the endocannabinoid anandamide are reduced in patients with untreated newly diagnosed temporal lobe epilepsy. Epilepsia. 2010 May;51(5):768-72.

----------

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

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

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

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

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

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

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

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

----------

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

[2] Boga JA. et al. Beneficial actions of melatonin in the management of viral infections: a new use for this "molecular handyman"? Rev Med Virol. 2012 Sep;22(5):323-38.

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

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

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

[6] de Magistris L. et al. Alterations of the intestinal barrier in patients with autism spectrum disorders and in their first-degree relatives. J Pediatr Gastroenterol Nutr. 2010 Oct;51(4):418-24.

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

----------

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

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

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

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

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

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

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

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

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

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

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

----------

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

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

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

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

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

----------

"patients with CFS/ME do not exhibit insufficient concentrations of circulating total 25(OH)D"

The title heading this post comes from the findings reported by Kate Earl and colleagues [1] (open-access available here), where '25(OH)D' refers to calcifediol, a compound typically used to estimate how much vitamin D is present in the body and CFS/ME refers to Chronic Fatigue Syndrome / Myalgic Encephalomyelitis.

After assaying some 92 people with CFS/ME and an almost equal number of 'age-matched healthy controls' (HCs) for plasma total 25(OH)D and individual vitamin D metabolites - "25(OH)D2 and 25(OH)D3" - researchers concluded that vitamin D deficiency was not rife in their cohort. Indeed we are told that: "total 25(OH)D was significantly higher (p=0.001) in serum of patients with CFS/ME compared with HCs (60.2 and 47.3 nmol/L, respectively)." The authors were also able to report that vitamin D supplementation by the CFS/ME group seemed to be a primary reason for their findings.

There are a few important strengths to the Earl results that are worth mentioning. Not least that vitamin D metabolites were measured by mass spectrometric methods similar to other independent research occasions (see here for example). Mass spectrometry seems to have quite a few advantages over other methods of vitamin D analysis; now labelled as a gold-standard technique. Added to their use of a deuterated standard ("hexadeuterated (OH)D3") and one has some degree of confidence in the analytical results; albeit, as the authors acknowledge: "that only the main marker of vitamin D status, that is, 25(OH)D, was measured" and how "there is a need to assess all of the vitamin D metabolites" of which there are quite a few [2].

At first glance, the Earl findings seem pretty unremarkable. Supplementation with vitamin D, as everyone is being encouraged to do these days (see here), means higher levels of circulating vitamin D. I would be surprised if they didn't. This is also not the first time that vitamin D levels in relation to CFS/ME have been talked about in the peer-reviewed domain either (see here) albeit not always with the same results but again with that caveat about supplementation in mind.

Given however that a measure of fatigue - "the Chalder Fatigue Questionnaire" - was also included for all participants in the Earl study, and how nothing very much seemed to be present when looking at any connection between fatigue scores and vitamin D status, this provides a possible clue that vitamin D is probably not a big player specifically in relation to the presentation of fatigue in most cases of CFS/ME. Such a questionnaire does not rule out other potential associations (e.g. post-exertional malaise, PEM) nor that other, potential comorbidity appearing alongside CFS/ME might not have a stronger vitamin D link (see here for example). But for now, it adds to the literature (see here) casting doubt on any direct role for vitamin D in relation to CFS/ME.

----------

[1] Earl KE. et al. Vitamin D status in chronic fatigue syndrome/myalgic encephalomyelitis: a cohort study from the North-West of England. BMJ Open. 2017 Nov 8;7(11):e015296.

[2] Abu Kassim NS. et al. Simultaneous determination of 12 vitamin D compounds in human serum using online sample preparation and liquid chromatography-tandem mass spectrometry. J Chromatogr A. 2017 Dec 6. pii: S0021-9673(17)31772-7.

----------