Monday, 28 April 2014

The best books... are those that tell you what you know already

The title for this post comes from a quote in George Orwell's Nineteen Eighty-Four. I'm using it to plug the fact that as from today - Monday 28th April 2014 - my book is out.

'Autism: Exploring the Benefits of a Gluten- and Casein-Free Diet. A practical guide for families and professionals' is available from the publishers site (Routledge) and lots of other real and virtual bookstores. I'm pretty sure that the book title is self-explanatory but just in case you need more information about it, here is the accompanying blurb on what we've tried to do:

"Once considered a rare condition, increasing numbers of children are being diagnosed as being on the autism spectrum, making it one of the most common and often challenging childhood developmental diagnoses. Whilst experts around the globe strive to unravel and truly understand how autism develops and presents itself, comparatively little is still known about the condition despite decades of research and investigation.

What is becoming clear is that autism is an extremely diverse condition with a presentation often complicated by a heightened risk of various accompanying behaviours and conditions outside of autism, variably affecting quality of life and future outcomes. Based on years of experimental study and thousands of personal observations, a growing body of research suggests that some challenging characteristics present in autism may be positively affected by the introduction of a gluten- and casein-free (GFCF) diet.

Autism: Exploring the Benefits of a Gluten- and Casein-Free Diet offers parents, teachers, and other education or health professionals with an easy-to-read alternative to sifting through the combined science. Written by leading experts in autism research, food, nutrition and dietetics, the book cuts through the jargon to offer readers a no-nonsense, accessible and authoritative overview of how diet might affect some characteristics of autism, and provides a range of useful recipes and handy hints for making mealtimes fun for children with autism and related conditions who are embarking on such a dietary change".

OK, so 'leading expert' might be a bit OTT for my part. Still, my co-authors and I have tried to provide a balanced overview of what the GFCF diet is and importantly what it isn't and where we're up to research-wise. GFCF recipes y'say? Yes, we've also put a few of them in the book too. Enjoy!

Sunday, 27 April 2014

MMP-9 and symptom severity of ADHD

"The statistical regression analysis revealed a correlation between increased serum MMP-9 levels and severity of symptoms in the ADHD". That was the sentence that caught my eye taken from the paper by Halina Kadziela-Olech and colleagues [1] (open-access here) looking at serum matrix metalloproteinase-9 (MMP-9) levels in children with hyperkinetic disorder (HKD) [2] "a severe form of a syndrome which is referred to in DSM-IV... and the American literature as attention deficit hyperactivity disorder (ADHD)".
Hamlet @ Wikipedia 

MMP-9 is something that has already been talked about on this blog with autism in mind (see here) but basically refers to a protease which is involved in degrading proteins of the extracellular matrix [3] and other duties. As Kadziela-Olech et al  point out, there is some preliminary interest in MMP-9 with reference to "neuropsychiatric disorders" particularly with schizophrenia in mind [4]. Interestingly also is that quite a bit of the conversation about MMP-9 and its potential effects have focused on the blood-brain barrier [5] under certain circumstances, which is something that I've always been more than a little interested in (see here) particularly transport across this important barrier.

Anyhow, back to the paper in question:

  • Analysis was conducted on 37 boys with HKD/ADHD all properly assessed, as were cognitive functions (via the WISC-R). Blood samples were donated and MMP-9 activity assessed by ELISA (see here).
  • Results: bearing in mind there was no asymptomatic control group, it's pretty pointless for me to talk about the serum levels of detected MMP-9 in cases overall. Of more relevance were the various associations made between levels of MMP-9 and presented symptoms: "The MMP-9 levels were significantly associated with symptoms severity of HKD and of ADHD".
  • In particular, MMP-9 levels seemed to correlate best with the impulsivity symptom domain when it came to HKD.

In the paper discussion, the authors do go through some of the possible ways that MMP-9 might relate to ADHD/HKD. Part of those discussions focus on blood-brain barrier (BBB) permeability and whether some of the reported risk factors for ADHD may affect MMP-9 expression onwards to a sort of 'leaky' BBB (sounds very familiar). I note also mention of minocycline as a MMP-9 inhibitor (see here) which may very well tie back into some of the research work being done with autism / Fragile X syndrome in mind, particularly given the overlap being reported between diagnoses like autism and ADHD. Some light reading around the topic of MMP-9 also reveals that there may be lots of other functions/relationships to had for this protease. With my cherry-picking hat on, I note the paper by Rodrigues and colleagues [6] talking about inflammation and in particular: "MMP-9 expression in the colon causes alterations in the fecal microbiome" given my interest in all-things gut microbiomics.

Ultimately however, quite a bit more work needs to be done in this area before anyone gets too carried away. Bearing in mind the Kadziela-Olech study was a snapshot study and as I said, without relevant control groups, there are some limitations on what can be said about the findings. Looking also at the plots correlating behavioural presentation and MMP-9 levels it's quite obvious that the relationship between the two variables is not altogether straight-forward despite what the statistics might suggest.

But don't let that deflect from their findings and the need for more research in this area...


[1] Kadziela-Olech H. et al. Serum matrix metalloproteinase-9 levels and severity of symptoms in boys with attention deficit hyperactivity disorder ADHD/hyperkinetic disorder HKD. Eur Child Adolesc Psychiatry. 2014 Mar 17.

[2] Cameron M. & Hill P. Hyperkinetic Disorder: Assessment and Treatment. Adv Psychiatr Treat. 1996; 2: 94-102.

[3] Birkedal-Hansen H. et al. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4(2):197-250

[4] Yamamori H. et al. Plasma levels of mature brain-derived neurotrophic factor (BDNF) and matrix metalloproteinase-9 (MMP-9) in treatment-resistant schizophrenia treated with clozapine. Neurosci Lett. 2013 Nov 27;556:37-41.

[5] Li YJ. et al. Disruption of the blood-brain barrier after generalized tonic-clonic seizures correlates with cerebrospinal fluid MMP-9 levels. J Neuroinflammation. 2013 Jul 5;10:80.

[6] Rodrigues DM. et al. Matrix metalloproteinase 9 contributes to gut microbe homeostasis in a model of infectious colitis. BMC Microbiol. 2012 Jun 13;12:105.

---------- Kadziela-Olech H, Cichocki P, Chwiesko J, Konstantynowicz J, & Braszko JJ (2014). Serum matrix metalloproteinase-9 levels and severity of symptoms in boys with attention deficit hyperactivity disorder ADHD/hyperkinetic disorder HKD. European child & adolescent psychiatry PMID: 24633733

Friday, 25 April 2014

Maternal thyroid dysfunction and autism or ADHD?

The paper by Andersen and colleagues [1] talking about the risk of offspring attention deficit hyperactivity disorder (ADHD) and/or autism spectrum disorder (ASD) where mothers presented with "maternal thyroid disease" is the source material for this post.
The Circus @ Wikipedia 

Based on data derived from Denmark, and those very useful Danish health registries which seem to be producing all-manner of potentially important information (see here), researchers reported that: "Maternal hyperthyroidism diagnosed and treated for the first time after the birth of the child increased the risk of ADHD in the child (adjusted HR 1.23; 95% CI 1.05-1.44), whereas hypothyroidism increased the risk of ASD (adjusted HR 1.34; 95% CI 1.14-1.59)".

Upon reading the suggested correlation between maternal hypothyroidism - where thyroid function is reduced - and something of an increased risk for an autism diagnosis in offspring, my mind headed back to the study by Gustavo Román and colleagues [2] talked about in this post, and their findings of "a consistent association between severe, early gestation maternal hypothyroxinemia and autistic symptoms in offspring". Of course, there are some differences between the way the Anderson and Román studies were conducted (registry diagnosis of thyroid dysfunction post-pregnancy vs. laboratory testing of thyroid function during early pregnancy) but the similar direction of the findings with autism in mind is interesting.

Equally interesting was the suggestion that maternal hyperthyroidism - an overactive thyroid - was related to some heightened risk of offspring ADHD in the Anderson cohort. A quick trawl of the limited research carried out in this area reveals some support for this assertion as per the data produced by Päkkilä and colleagues [3] who reported: "Increases in maternal TSH [thyroid stimulating hormone] in early pregnancy showed weak but significant association with girls' ADHD symptoms". That being said, I don't doubt that this is going to be a complicated relationship as per other reports on things like maternal thyroid autoimmunity during pregnancy and offspring outcomes [4] and the issue of iodine and offspring outcome [5] which has previously been talked about on this blog with autism in mind (see here). The fact that autism and ADHD can and do present comorbid in quite a few people adds to the complication.

I'd be hard-pressed to offer anything definitive about how and why thyroid function might impact specifically on the risk of diagnoses such as ADHD and autism outside of what is already known about maternal thyroid function and infants (see here). I assume as part of a larger clinical picture, there may be some direct effects specifically acting on the infant during the nine months that made us as well as other peripheral effects acting on for example, delivery issues as per the correlation between thyroid function and something like preterm birth risk [6] which might play some role [7].

Similarly when talking about the 'hows' of how issues with thyroid function come about in mums-to-be, one is faced with an array of possible factors. I could start going on about some of the genetics of thyroid disease [8] but I have neither the time, space or expertise to provide any thorough overview. Environment can also play a role as per the factors listed on this website so take your pick as to what might be relevant or not to cases. With the continuing interest in environmental effects and conditions like autism and ADHD, I might direct you to some of the work done on endocrine disruptors and thyroid functions [9] but with the realisation that the picture is going to be a complicated one varying from one group to the next, even one person to the next.

Still, the Anderson results further add to the interest looking at maternal thyroid function and offspring outcome and merit some further investigations in this area. Indeed, thyroid function is turning up in all manner of places these days [10] and perhaps point to some interesting associations which might have some important knock-on effects [11] ...

So, New Kids On The Block making a come-back.... You Got It!


[1] Andersen S. et al. Attention deficit hyperactivity disorder and autism spectrum disorder in children born to mothers with thyroid dysfunction: a Danish nationwide cohort study. BJOG. 2014 Mar 10.

[2] Román GC. et al. Association of gestational maternal hypothyroxinemia and increased autism risk. Ann Neurol. 2013 Nov;74(5):733-42.

[3] Päkkilä F. et al. The impact of gestational thyroid hormone concentrations on ADHD symptoms of the child. J Clin Endocrinol Metab. 2014 Jan;99(1):E1-8.

[4] Ghassabian A. et al. Maternal thyroid autoimmunity during pregnancy and the risk of attention deficit/hyperactivity problems in children: the Generation R Study. Thyroid. 2012 Feb;22(2):178-86.

[5] Vermiglio F. et al. Attention deficit and hyperactivity disorders in the offspring of mothers exposed to mild-moderate iodine deficiency: a possible novel iodine deficiency disorder in developed countries. J Clin Endocrinol Metab. 2004 Dec;89(12):6054-60.

[6] Stagnaro-Green A. et al. The thyroid and pregnancy: a novel risk factor for very preterm delivery. Thyroid. 2005 Apr;15(4):351-7.

[7] Silva D. et al. Environmental risk factors by gender associated with attention-deficit/hyperactivity disorder. Pediatrics. 2014 Jan;133(1):e14-22.

[8] Medici M. et al. Identification of novel genetic Loci associated with thyroid peroxidase antibodies and clinical thyroid disease. PLoS Genet. 2014 Feb 27;10(2):e1004123.

[9] Hofmann PJ. et al. Interference of endocrine disrupters with thyroid hormone receptor-dependent transactivation. Toxicol Sci. 2009 Jul;110(1):125-37.

[10] Wysokiński A. & Kłoszewska I. Level of Thyroid-Stimulating Hormone (TSH) in Patients with Acute Schizophrenia, Unipolar Depression or Bipolar Disorder. Neurochem Res. 2014 Apr 11.

[11] van der Pals M. et al. Prevalence of thyroid autoimmunity in children with celiac disease compared to healthy 12-year olds. Autoimmune Dis. 2014;2014:417356.

---------- Andersen S, Laurberg P, Wu C, & Olsen J (2014). Attention deficit hyperactivity disorder and autism spectrum disorder in children born to mothers with thyroid dysfunction: a Danish nationwide cohort study. BJOG : an international journal of obstetrics and gynaecology PMID: 24605987

Thursday, 24 April 2014

As if you needed telling...

"GI [gastrointestinal] dysfunction was prevalent in this cohort of children with ASD [autism spectrum disorders], observations consistent with the reports of parents and other clinicians". That was one of the conclusions reached by Victor Kang and colleagues [1] in their study looking at GI issues in cases of autism.

Of course we've been here before... many times in fact, as autism research delivers more evidence that bowel issues are quite frequently over-represented in cases of autism (see here and see here and see here). That the authors also conclude: "GI dysfunction in ASD requires proper evaluation and treatment" is the next stage in the evolution of this research area despite the fact that recommendations already exist [2] and onwards the question of whether treating said bowel issues might impact on presented core or peripheral signs and symptoms of autism. At the very least, whether dealing with bowel issues improves quality of life.

There's really no need for me to say too much more about the Kang results outside of one particular point when it came to the results of the "endoscopic and colonoscopic evaluations" carried out on a small percentage of study participants involved in this work. To quote: "Inflammation of the gut was found in 6 of the 12 subjects" meaning that 50% of those examined carried some potential issue with the GI tract. Of course this is not the first time that such findings have been reported (see here and see here) and I very much doubt it will be the last either. I'm drawn back to the Stephen Walker findings (see here) as one example of where autism research might want to continue to venture when it comes to inflammatory bowel disease (if that's what I can call it) and autism, but will say little more than that at this time.

And finally, I know that endoscopic evaluations are hardly desirable for anyone, which is why we should also be investing quite a bit more research into alternatives such as the wireless capsule endoscopy which has already seen some use in the area of autism research [3].


Update: 28/04/14. And like waiting for buses, they all come along at once as per the meta-analysis of GI issues in autism research by Barbara McElhanon and colleagues [4] (open-access here) concluding a: "greater prevalence of GI symptoms among children with ASD compared with control children". Need I say more?


[1] Kang V. et al. Gastrointestinal Dysfunction in Children With Autism Spectrum Disorders. Autism Res. 2014 Apr 21. doi: 10.1002/aur.1386.

[2] Buie T. et al. Recommendations for evaluation and treatment of common gastrointestinal problems in children with ASDs. Pediatrics. 2010 Jan;125 Suppl 1:S19-29.

[3] Balzola F. et al. Panenteric IBD-like disease in a patient with regressive autism shown for the first time by the wireless capsule enteroscopy: another piece in the jigsaw of this gut-brain syndrome? Am J Gastroenterol. 2005 Apr;100(4):979-81.

[4] McElhanon BO. et al. Gastrointestinal Symptoms in Autism Spectrum Disorder: A Meta-analysis. Pediatrics. 2014. April 28.

---------- Kang V, Wagner GC, & Ming X (2014). Gastrointestinal Dysfunction in Children With Autism Spectrum Disorders. Autism research : official journal of the International Society for Autism Research PMID: 24753336

Wednesday, 23 April 2014

Phenylalanine and schizophrenia: new directions for intervention?

As regular readers might already have noticed, amino acids are a bit of a obsession of mine on this blog. Out of all of them - and there are quite a few - I'm particularly interested in the aromatic amino acids and the their various connections to health and wellbeing. I've talked at length about some of the proposed connections made between amino acids such as tryptophan, tyrosine and phenylalanine to all manner of conditions but specifically with the autism spectrum in mind (see here).
The conversion. Matthews (2007) J Nutr. 137: 15495-15555.

Phenylalanine (or Phe) has been a particular favourite on this blog, not least because of its connection to that most classical 'diet can affect mental health' condition known as Phenylketonuria (PKU). As per other research chatter however, the connection between phenylalanine and PKU might just be the tip of the iceberg (see here). Indeed, today that iceberg just got a little bigger as I discuss the paper by Olaoluwa Okusaga and colleagues* (open-access) and their observations of elevated blood levels of phenylalanine in cases of schizophrenia. Such findings might indeed have some important management consequences as you'll see shortly when it comes to the use of something called BH4.

The Okusaga paper is open-access but a few of the important details:

  • Well, one can't say that this was an under-powered study from a participant number point of view, as blood samples from 950 adult participants with a confirmed diagnosis of schizophrenia via the SCID were compared with 1000 asymptomatic controls for levels of phenylalanine and tyrosine.
  • Analysis of samples was via HPLC with fluorescence detection, which whilst OK as a separative-detection method is not exactly the gold-standard that is mass spectrometry (MS) or nuclear magnetic resonance (NMR)
  • From the measures of phenylalanine and tyrosine, an estimate of the activity of phenylalanine hydroxylase (PAH) was also calculated and expressed as a phenylalanine: tyrosine ratio**. PAH represents an important step in the conversion of phenylalanine to tyrosine, which then proceeds down a metabolic pathway to eventually end up as dopamine. It's worth pointing out that dopamine has some important research history when it comes to the presentation of schizophrenia or at least, that's the suggestion (see here).
  • Results: well bearing in mind some issues with the matching of the two sample groups in terms of age and BMI (a higher BMI in the schizophrenia group bearing in mind that these were not medication-naive participants), the schizophrenia group "had significantly higher Phe (geometric mean difference 1.26 µmol/L; CI 1.18 to 1.36, p<0.0001) and Phe:Tyr ratio (geometric mean difference 1.41; CI 1.33 to 1.48, p<0.0001) compared to healthy controls and this finding persisted after controlling for gender, age, education, and BMI differences between the 2 groups". As a group however, there was no significant differences for the schizophrenia and control groups when it came to measures of tyrosine although "lower levels of Tyr are more common among schizophrenia patients".
  • The authors conclude that alongside further, more controlled study with regards to sample collection (including looking at measures of inflammation), there may also be some merit in looking at the potential effects of "Phe-lowering interventions in schizophrenia".

As I mentioned before, Phe-lowering interventions very much includes the use of BH4, but could also mean the rather more invasive use of a low phenylalanine diet (and then tyrosine supplements?) more commonly indicated for cases of PKU. I should point out that this does not mean I am in any way endorsing such a dietary change or pharmacological action at this time as a function of my caveat on this blog about not giving medical or clinical advice. That being said, the research gauntlet has been thrown down by the results of the Okusaga study so I'll be keeping my eyes open for future work in this area. 

There is other evidence suggestive of issues with the availability of BH4 in cases of schizophrenia as per the results from Richardson and colleagues*** which also extended to related schizoaffective disorder too****. Given that BH4 provides an important support service to a variety of enzymes relevant to the metabolism of aromatic amino acids (think tryptophan hydroxylase, TPH, for example), lower levels are probably not all that desirable. This might be particularly important to ensuring phenylalanine does not build up to too higher levels and the effects that can have*****.

Aside from the phenylalanine-lowering interventions call made from the Okusaga study, a few other questions are floating round my mind. So, at what point do phenylalanine levels become elevated in some cases of schizophrenia? I'd assume that as per the quite comprehensive use of the Guthrie test these days, we aren't talking about participants reaching the cut-off points for PKU in early infancy, so when does this issue present itself in cases of schizophrenia and why? I'm also interested in the cognitive effects of [chronic] elevated phenylalanine levels and how this might also map onto similar elevations noted in cases of schizophrenia too. Noting the increasing interest in cognition and schizophrenia (see this paper****** for example) and the growing  importance of cognitive impairment to cases, could the elevated phenylalanine results merely reflect this one facet of schizophrenia?

So you can see that there is a lot more to do in this area. Given also that schizophrenia, like autism, is probably better represented on a spectrum model, the question is also whether hyperphenylalaninemia in relation to cases of schizophrenia might represent one particular part of that schizophrenia spectrum? At least one other study suggests possibly******* (open-access) with quite a novel alternative method for detecting phenylalanine used. A lot more to do in this area methinks.

Music to close. Driftwood by Travis.


* Okusaga O. et al. Elevated Levels of Plasma Phenylalanine in Schizophrenia: A Guanosine Triphosphate Cyclohydrolase-1 Metabolic Pathway Abnormality? PLoS ONE 2014. 9(1): e85945.

** Matthews DE. An Overview of Phenylalanine and Tyrosine Kinetics in Humans. J. Nutr. 2007; 137: 15495-15555.

*** Richardson MA. et al. Evidence for a tetrahydrobiopterin deficit in schizophrenia. Neuropsychobiology. 2005;52(4):190-201.

**** Richardson MA. et al. Analysis of plasma biopterin levels in psychiatric disorders suggests a common BH4 deficit in schizophrenia and schizoaffective disorder. Neurochem Res. 2007 Jan;32(1):107-13.

***** Pascucci T. et al. Behavioral and neurochemical characterization of new mouse model of hyperphenylalaninemia. PLoS One. 2013 Dec 20;8(12):e84697.

****** Keefe RS. & Harvey PD. Cognitive impairment in schizophrenia. Handb Exp Pharmacol. 2012;(213):11-37.

******* Teraishi T. et al. 13C-phenylalanine breath test detects altered phenylalanine kinetics in schizophrenia patients. Translational Psychiatry 2012; 2: e119.

---------- Olaoluwa Okusaga, Olesja Muravitskaja, Dietmar Fuchs, Ayesha Ashraf, Sarah Hinman, Ina Giegling, Annette M. Hartmann, Bettina Konte, Marion Friedl, Jason Schiffman, Elliot Hong, Gloria Reeves, & et al (2014). Elevated Levels of Plasma Phenylalanine in Schizophrenia: A Guanosine Triphosphate Cyclohydrolase-1 Metabolic Pathway Abnormality? PLoS ONE, 9 DOI: 10.1371/journal.pone.0085945

Monday, 21 April 2014

Lathosterolosis, cholesterol and autism?

Although intrigued by the findings reported by Pier Luigi Calvo and colleagues [1] describing a "unique case" potentially linking liver functions and cognitive functions with a hat-tip to the presentation of autistic behaviours, I'll readily admit that I am way out of my comfort and competence zones when discussing this paper so please be ready with that pinch of salt.

How do you like your eggs in the morning? @ Wikipedia 
As per what the paper and accompanying press release (see here) indicate, this was a case report of a young girl diagnosed with lathosterolosis [2] - a rare inborn error of metabolism characterised by the accumulation of 5α-Cholest-7-en-3β-ol (lathosterol), an intermediate compound tied into the metabolism of cholesterol - and her changing clinical presentation following a liver transplant. The authors note that this was the "only surviving patient with lathosterolosis" such is the seriousness of the condition.

In case you want the real heavy biochemistry, have a look at the paper by Brunetti-Pierri and colleagues [3] discussing all-things lathosterolosis and in particular, the crucial part played by the enzyme 3-beta-hydroxysteroid-delta-5-desaturase (sterol-C5-desaturase or SC5D) in the condition.

The suggestion from the Calvo study that "timely liver transplantation might arrest the progression of neurological damage caused by diseases related to problems with cholesterol production" whilst sounding pretty invasive is nonetheless, of potential importance to a wider area of research. The details of outcome describing "a complete biochemical recovery, an arrest of mental deterioration and a stable MRI picture" at 5-year follow-up are nothing short of remarkable.

As far as I can make out, quite a bit of the discussion where autism or autistic behaviours is mentioned revolves around the correlation between issues with social interaction, exploration of her environment and cholesterol biosynthesis, and what happened when cholesterol levels were normalised as a consequence of the liver transplant. Certainly this is not the first time that cholesterol and autism have been mentioned in the same breath together. In a post going back some years now, I talked about the various research in this area, and specifically a condition called Smith-Lemi Opitz syndrome (SLOS) which represents the prototypical 'issues with cholesterol formation can affect physiology and behaviour' condition. The paper by Sikora and colleagues [4] titled: 'The near universal presence of autism spectrum disorders in children with Smith-Lemli-Opitz syndrome' kinda hinted at the link between SLOS and the presentation of autism/autistic behaviours, compounded by other data [5] (open-access here) correlating low cholesterol levels and autism in some cases. Remember: autisms not autism.

Just in case anyone gets the wrong idea about my interpretation of this research, I'm not for one minute suggesting that a liver transplant is indicated for cases of autism outside of when medically required. As the study author notes: "We were dealing with a unique case—literally, as the child is the only known surviving patient with the condition—so it is difficult drawing inferences of broader significance". That however the liver provides an indispensable array of functions (see here too) might however mean that any whole body research approach applied to autism (not just looking at the grey/pinkish matter floating in the skull) might include some basic analysis of it's function too. As noted from other research in this area such as that included in the paper by Horvath & Perman [4] discussing sulphation (sulfation) capacity of the liver and cases of autism, there is potentially more to do in this area. Other work by Wakefield and colleagues [6] (open-access here) suggesting that: "hepatic encephalopathy represents a prototypic afferent gut–brain interaction that may provide insight into other encephalopathic states that have been linked to extra-cranial pathology", might also provide some food for thought in this area too.

Finally, continuing the possibility of a cholesterol connection to some autisms, I'll refer you back to some very interesting data talked about on the SFARI blog last year (2013) on the possibility of statins and cholesterol supplements as areas of future investigation (with no medical or clinical advice given or intended). A hat-tip also goes to Peter over at the Epiphany blog and his series of articles on statins and autism too with some accompanying information about side-effects too [7].

Music to close. And after recently watching the documentary on the reformation of the Stone Roses, a pretty famous song... Waterfall and memories of growing up listening to the Stone Roses the first time around come flooding back. 


[1] Calvo PL. et al. Liver transplantation in defects of cholesterol biosynthesis: the case of lathosterolosis. Am J Transplant. 2014. March 12.

[2] Krakowiak PA. et al. Lathosterolosis: an inborn error of human and murine cholesterol synthesis due to lathosterol 5-desaturase deficiency. Hum Mol Genet. 2003 Jul 1;12(13):1631-41.

[3] Brunetti-Pierri N. et al. Lathosterolosis, a Novel Multiple-Malformation/Mental Retardation Syndrome Due to Deficiency of 3β-Hydroxysteroid-Δ5-Desaturase. Am J Hum Genet. Oct 2002; 71(4): 952–958.

[4] Sikora DM. et al. The near universal presence of autism spectrum disorders in children with Smith-Lemli-Opitz syndrome. Am J Med Genet A. 2006 Jul 15;140(14):1511-8.

[5] Horvath K. & Perman JA. Autism and gastrointestinal symptoms. Curr Gastroenterol Rep. 2002 Jun;4(3):251-8.

[6] Wakefield AJ. et al. Review article: the concept of entero-colonic encephalopathy, autism and opioid receptor ligands. Aliment Pharmacol Ther. 2002 Apr;16(4):663-74.

[7] Finegold JA. et al. What proportion of symptomatic side effects in patients taking statins are genuinely caused by the drug? Systematic review of randomized placebo-controlled trials to aid individual patient choice. Euro J Preventive Cardiology. 2014;  March 12.

---------- Calvo, P., Brunati, A., Spada, M., Romagnoli, R., Corso, G., Parenti, G., Rossi, M., Baldi, M., Carbonaro, G., David, E., Pucci, A., Amoroso, A., & Salizzoni, M. (2014). Liver Transplantation in Defects of Cholesterol Biosynthesis: The Case of Lathosterolosis American Journal of Transplantation DOI: 10.1111/ajt.12645

Thursday, 17 April 2014

Mitochondrial dysfunction as a neurobiological subtype of autism

The paper by Suzanne Goh and colleagues [1] reporting on "a possible neurobiological subtype of mitochondrial dysfunction in ASD [autism spectrum disorder]" is a worthy addition to the research roll call which has graced this blog down the years. Based on the analysis of brain lactate levels - a potential marker of mitochondrial dysfunction - via the analysis of lactate doublets on brain magnetic resonance spectroscopic imaging (MRSI), authors picked up a significantly higher rate of lactate in cases of autism spectrum disorder (ASD) when compared to age and sex-matched asymptomatic controls. I've talked lactate and autism before on this blog (see here) so very much welcomed this research looking specifically at brain levels of this stuff.

I'm writing this post having already scheduled a blog entry on the recent paper by Rose and colleagues [2] (open-access here) on the increasing complexity of mitochondrial dysfunction being seemingly present in some cases of autism. Given the findings from Goh et al I've decided to publish this entry first (just to confuse everyone even further) as yet again, my confusion on the topic of all-things mitochondrial has an opportunity to shine through.

So then, a few details from the Goh paper:

  • Based on imaging and other data derived from 75 participants diagnosed with an ASD (aged 5-60 years) contrasted with 96 typically-developing controls, the authors set about "assessing in-vivo evidence of mitochondrial dysfunction directly in the brains of a large sample of children and adults with ASD".
  • Whilst not an imaging man, I can tell you that they used proton multiplanar spectroscopic imaging (MPSI) to quantify endogenous brain chemistry and "regional cellular metabolism and function" specifically towards the detection of lactate. Actually, the talk of [lactate] doublets is not a million miles away from the results one gets as a consequence of a related chemical analytical technique, NMR, which brings back memories of some work from days gone by.
  • After laying down quite a few ground rules for what was and wasn't a readable result, the authors concluded that: "Lactate doublets were present at a significantly higher rate in participants with ASD (13%) than in typically developing controls (1%) (P = .001), providing in vivo evidence for the presence of mitochondrial dysfunction in the brains of individuals with ASD". In-vivo by the way, means in the living and contrasts with science done in a test-tube (in-vitro).
  • Age was a factor when it came to lactate levels, with elevations reported more often in adults than in children. This phenomenon has been talked about before in the research literature [3].
  • The authors go on to discuss the implications of their results. Bearing in mind the various situations where elevated brain lactate levels have been noted outside of just ageing, including as a result of issues like anxiety or panic disorder [4], they reiterate how their "strict exclusion critera and careful scanning procedures made such explanations less likely". Further they highlight how: "individuals with ASD should undergo evaluation for mitochondrial dysfunction, as novel and promising treatments are under development for mitochondrial disorders".

As per my link above, this is not the first time that lactate has appeared in the autism research literature. I'll for example, draw your attention to the paper by Al-Mosalem and colleagues [4] and their reporting that: "Lactate as an important energy metabolite for the brain was significantly higher in autistic patients compared to control showing about 40% increase". Bear in mind however that this and other results [5] have tended to look in plasma rather than directly what's going on in the brain as Goh et al did.

There's little more for me to say on this area of research aside from the need for further replicative investigations and perhaps a little more inquiry into the subgroup of people with autism who fall into this mitochondrial dysfunction category bearing in mind the continued focus on the plurality of autism (the autisms). That there may be interventions available for mitochondrial disorder when present [6] is another important point. As per related research in other conditions with a potential mitochondrial aspect to them (see here), at least one of the interventions - Coenzyme Q10 (ubiquinol) - is being looked at with some autism in mind [7] (open-access here) bearing in mind no medical or clinical advice is given or intended.

Music then to close. I'm thinkin' of something with a candy orientation given the time of year, so again, ladies and gentlemen, Mr Sammy Davis Jnr and The Candy Man.. (he can you know).


[1] Goh S. et al. Mitochondrial Dysfunction as a Neurobiological Subtype of Autism Spectrum Disorder. Evidence From Brain Imaging. JAMA Psychiatry. 2014. April 9.

[2] Rose S. et al. Oxidative stress induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell lines. Transl Psychiatry. 2014 Apr 1;4:e377.

[3] Ross JM. et al. High brain lactate is a hallmark of aging and caused by a shift in the lactate dehydrogenase A/B ratio. PNAS. 2010; 10.1073/pnas.1008189107

[4] Al-Mosalem OA. et al. Metabolic biomarkers related to energy metabolism in Saudi autistic children. Clin Biochem. 2009 Jul;42(10-11):949-57.

[5] Oliveira G. et al. Mitochondrial dysfunction in autism spectrum disorders: a population-based study. Dev Med Child Neurol. 2005 Mar;47(3):185-9.

[6] Parikh S. et al. A Modern Approach to the Treatment of Mitochondrial Disease. Curr Treat Options Neurol. Nov 2009; 11(6): 414–430.

[7] Gvozdjáková A. et al. Ubiquinol improves symptoms in children with autism. Oxid Med Cell Longev. 2014;2014:798957.

---------- Goh, S., Dong, Z., Zhang, Y., DiMauro, S., & Peterson, B. (2014). Mitochondrial Dysfunction as a Neurobiological Subtype of Autism Spectrum Disorder JAMA Psychiatry DOI: 10.1001/jamapsychiatry.2014.179

Wednesday, 16 April 2014

Joined by HDAC (inhibitors)

I'm treading quite carefully with this post which came about following my [non-expert] reading of the paper abstract from Anand Venkatraman and colleagues [1] on a potential downside to the use of HDAC (histone deacetylase) inhibitors for treating spinocerebellar ataxia type 1 (SCA1), a progressive disease affecting movement and other knock-on functions. This follows other work suggesting that certain HDAC inhibitors might offer some important new lines of investigation when it comes to at least some of the various types of spinocerebellar ataxia (SCA). For those who thought this was a blog about autism research, bear with me on this one...

HDACs represent a group of enzymes which go to work removing acetyl groups on histone tails which, as the paper by Patrick Grant [2] (open-access) very nicely illustrates, has the potential to do some rather important things to processes like gene expression (condensing chromatin and repressing transcription). I have kinda touched upon histones and the so-called histone code in a previous introductory post on the rise and rise of epigenetics (see here) with autism in mind.

The Venkatraman results focused on a mouse model, and how depletion/loss of a particular type of HDAC - HDAC3 - was in some cases: "highly deleterious both behaviorally, with mice showing early onset ataxia, and pathologically, with progressive histologic evidence of degeneration". They talk about "cautionary evidence that this approach could produce untoward effects" when it comes to the employment of "pharmacologic inhibition of HDAC3" via HDAC inhibitors in SCA.

Not to make too many sweeping generalisations or form associations which might not be there, but two things from the Venkatraman paper got my old(ish) grey matter fired up: (i) mention of HDAC inhibitors and the emerging story when it comes to prenatal exposure to valproate with a HDAC slant, and (ii) the focus on Purkinje cell function; as their paper title states: "The histone deacetylase HDAC3 is essential for Purkinje cell function". Both these points bring me back to some potentially important issues which might apply to at least some autism and related neurodevelopmental outcomes.

It is still very much an emerging picture but pregnancy use of valproate and 'adverse' offspring events/development is turning into something of a quite important association in recent times. So much so that the US FDA and UK MHRA have issued some guidance on this matter. Valproate has some history as a potential teratogen [3] bearing in mind my offering no medical or clinical advice on this matter aside from saying 'don't mess with epilepsy'. That valproate is also an HDAC inhibitor [4] (open-access) is another mechanism through which the drug might (a) find some new markets for conditions other than epilepsy, but also (b) impact on development and functions. Readers are invited to have a look through the paper by Katie Lloyd [5] (open-access) for a well-rounded overview of potential effects.

Then to the Purkinje cell story. I'm sure most people with an interest in autism will have heard about the cerebellum in relation to the condition at some point. Indeed, the paper by Fatemi and colleagues [6] (open-access) kinda sums up where we're at when it comes to the 'little brain' bearing in mind the need for further investigation and the greater focus on the plural 'autisms'. To talk about the cerebellum and autism also brings into the play those Purkinje cells which have also featured on several occasions on the autism research menu [7] and quite recently, with an epigenetic slant to the research (see here). Indeed, the paper by Jill James and colleagues [8] (open-access) on epigenetics and EN-2 is something I'd very much like to see more work on.

Again, not to make mountains out of molehills, but I did wonder whether there may be some science to do covering these potentially overlapping areas. I'm not necessarily saying that valproate = HDAC inhibition = impact on Purkinje cell numbers/maturation/functions = autism because I very much doubt it's going to be that simple or generalised despite some emerging [rodent] data [9]. With the increasing interest in all-things epigenetic however, also crossing over to autism research [10] (open-access), one might consider more inquiry into the HDACs, their inhibitors and effectors (and exposure timing) to be a potentially important part of that particular autism research tide? Whether even important ecosystems e.g. "the [gut] microbiota itself may be viewed as an epigenetic entity" [11] may also tie into some of the work in this area too?


[1] Venkatraman A. et al. The histone deacetylase HDAC3 is essential for Purkinje cell function, potentially complicating the use of HDAC inhibitors in SCA1. Hum Mol Genet. 2014 Mar 4.

[2] Grant PA. A tale of histone modifications. Genome Biology 2001, 2:reviews0003-reviews0003.6

[3] Alsdorf R. & Wyszynski DF. Teratogenicity of sodium valproate. Expert Opin Drug Saf. 2005 Mar;4(2):345-53.

[4] Göttlicher M. et al. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J. 2001; 20(24): 6969–6978.

[5] Lloyd KA. A scientific review: mechanisms of valproate-mediated teratogenesis. Bioscience Horizons 2013; 6 : hzt003

[6] Fatemi SH. et al. Consensus paper: pathological role of the cerebellum in autism. Cerebellum. 2012 Sep;11(3):777-807.

[7] Skefos J. et al. Regional alterations in purkinje cell density in patients with autism. PLoS One. 2014 Feb 24;9(2):e81255.

[8] James SJ. et al. Complex epigenetic regulation of engrailed-2 (EN-2) homeobox gene in the autism cerebellum. Transl Psychiatry. 2013 Feb 19;3:e232.

[9] Moldrich RX. et al. Inhibition of histone deacetylase in utero causes sociability deficits in postnatal mice. Behav Brain Res. 2013 Nov 15;257:253-64.

[10] Lasalle JM. Autism genes keep turning up chromatin. OA Autism. 2013 Jun 19;1(2):14.

[11] Stilling RM. et al. Microbial genes, brain & behaviour - epigenetic regulation of the gut-brain axis. Genes Brain Behav. 2014 Jan;13(1):69-86.

---------- Venkatraman A, Hu YS, Didonna A, Cvetanovic M, Krbanjevic A, Bilesimo P, & Opal P (2014). The histone deacetylase HDAC3 is essential for Purkinje cell function, potentially complicating the use of HDAC inhibitors in SCA1. Human molecular genetics PMID: 24594842

Monday, 14 April 2014

Neurology of inflammatory bowel diseases

The paper by Ben-Or and colleagues [1] talking about a neurologic profile present in a small participant cohort of children and adolescents diagnosed with an inflammatory bowel disease (IBD) caught my eye recently. Their findings reporting that over two-thirds of their paediatric participant group diagnosed with IBD also "exhibited neurologic manifestations" provides some compelling preliminary evidence for further investigation in this area.

Outside of reports of headache and dizziness, the presentation of attention-deficit hyperactivity disorder (ADHD), hypotonia and "sensory complaints" comorbid to IBD shines a spotlight on the so-called 'gut-brain axis'. That being said the fact that "seizures and neuropsychiatric disorders were less characteristic" between IBD cases and asymptomatic controls may also have some important implications for various conditions including the primary topic of this blog, the autism spectrum conditions.

I'm not on this occasion going to dissect the Ben-Or findings too much aside from pointing you in the direction of some other research which may very well tie into their findings. I've talked before about research on other bowel-related conditions suggestive of potentially important neurological and behavioural links. Think coeliac disease and the the ataxia story as one example mentioned in a previous post (see here). I'd also draw your attention to some work in the autism research domain talking about a possible link between functional bowel habit issues and the presentation of anxiety and sensory symptoms (see here). Granted, it is a leap from a diagnosis of IBD to the presentation of constipation or diarrhoea not necessarily due to an IBD (or at least that's what was thought) but one might imagine that further investigation would be indicated in light of the Ben-Or data.

Reiterating the gut-brain link which seems to be appearing with ever-greater frequency these days, I'm minded to suggest that further research be directed to looking at the possible mechanisms to account for any relationship. The usual triad of issues: intestinal (gut) permeability, the gut microbiota and the gut mucosal immune system spring to mind as potential players in any relationship, but that's all I'll say on the matter for now.


[1] Ben-Or O. et al. The Neurologic Profile of Children and Adolescents With Inflammatory Bowel Disease. J Child Neurol. 2014 Apr 2.

---------- Ben-Or O, Zelnik N, Shaoul R, Pacht A, & Lerner A (2014). The Neurologic Profile of Children and Adolescents With Inflammatory Bowel Disease. Journal of child neurology PMID: 24700662

Friday, 11 April 2014

Dad's obesity and risk of offspring autism

In this post I'm talking about the paper by Pål Surén and colleagues [1] (open-access here) and their suggestion that "paternal obesity is an independent risk factor for ASDs [autism spectrum disorders] in children". I do so not with the intent of stigmatising parents and specifically parents with weight issues, which tend to be present for many more reasons than just food and exercise (see here), but merely to highlight how parental physical health may show some relationship to offspring cognitive and developmental progress. Indeed, the findings from Surén et al are to viewed in the context of some other related research in this area (see here).
By the water @ Renoir @ Wikipedia 

Quite a nice summary of the Surén work can be found here and here. The basics are: MoBa (see here), questioning parents particularly fathers about their physical health while their partner was pregnant, following up offspring and their subsequent development - specifically whether they had received a diagnosis of "autistic disorder", Asperger syndrome or pervasive developmental disorder not otherwise specified (PDD-NOS).

The results: well, the rate of autism reported (0.45%) was interesting. Certainly compared to other prevalence estimates we've been hearing about recently, quite a different figure was noted altogether (see here) allowing for age differences in case ascertainment. Then to the primary findings: "The risk of autistic disorder was 0.27% (25 of 9267) in children of obese fathers and 0.14% (59 of 41 603) in children of fathers with normal weight (BMI <25), generating an adjusted OR of 1.73 (95% CI: 1.07–2.82)". And with regards to Asperger syndrome: "The risk was 0.38% (18 of 4761) in children of obese fathers and 0.18% (42 of 22 736) in children of normal-weight fathers, and the adjusted OR was 2.01 (95% CI: 1.13–3.57)". Ergo, potentially double the risk of offspring autism when fathers were categorised as overweight or obese based on body mass index (BMI).

It goes without saying that the suggestion of a link between paternal weight and offspring risk of autism is by no means proved by this latest research. Think correlation not being the same as causation as one reason why we should not just accept the results of this work at face value irrespective of how enthusiastic researchers might be about their data and what they were able to control for as potential interfering variables. That also BMI has it's 'issues' when it comes to defining healthy weight is another reason for caution where muscle mass for example, is not accounted for in such a simplistic formula.

But all that doesn't mean the results are not interesting...

In recent times I've noticed quite a bit more research looking at the potential role of father's health and wellbeing on offspring development treading in the footsteps of how ageing might play a role. Take for example the recent opinion piece in Nature titled 'Sins of the father' which introduces another side to the Surén results: the science of epigenetics. The paper by Lambrot and colleagues [2] is as good an example as any on how a father's nutritional status with folate in mind, might impact on offspring health. I'm not asking you to take this as fact; merely that the focus on maternal nutrition and offspring outcome might not be the only important variable in any relationship.

Harking back to another paper by the Surén research group (including Drs Hornig and Lipkin) [3] reveals nutrition to be something that the authors had probably thought about with the current results in mind. On that occasion, the focus was on the elevated risk of autism in cases of a short inter-pregnancy interval (see here) and mention of a "depletion of micronutrients" as a possible factor. Shadows indeed of the work of the late David Barker and his foetal programming hypothesis.

I do believe there are more investigation to be done building on the Surén results. I've already made mention of folate in the father-offspring relationship given the link between the folate cycle and the availability of methyl groups for the process of DNA methylation, an important epigenetic process. One might also wonder about the body of work looking at more traditional genetic issues in the genes involved in that cycle such as everyone's favourite Scrabble gene and enzyme:  Methylenetetrahydrofolate reductase (MTHFR) and its growing links with some autism (see here). Could issues with MTHFR present in fathers confer susceptibility to offspring autism, or weight issues pertinent to an elevated risk via epigenetic mechanisms? That being said, I'm sure that any relationship is going to be complicated and not necessarily relevant to every child diagnosed with an autism spectrum condition.

Music to close. In memory of author Sue Townsend and her Adrian Mole series of books... Profoundly in Love with Pandora.


[1] Surén P. et al. Parental Obesity and Risk of Autism Spectrum Disorder. Pediatrics. 2014. April 7.

[2] Lambrot R. et al. Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes. Nature Comms. 2013; 4: 2889.

[3] Gunnes N. et al. Interpregnancy interval and risk of autistic disorder. Epidemiology. 2013 Nov;24(6):906-12.

---------- Suren, P., Gunnes, N., Roth, C., Bresnahan, M., Hornig, M., Hirtz, D., Lie, K., Lipkin, W., Magnus, P., Reichborn-Kjennerud, T., Schjolberg, S., Susser, E., Oyen, A., Smith, G., & Stoltenberg, C. (2014). Parental Obesity and Risk of Autism Spectrum Disorder PEDIATRICS DOI: 10.1542/peds.2013-3664

Thursday, 10 April 2014

Gluten exposure and "feelings of depression"?

Could exposure to dietary gluten affect a person's moods or emotional state?

Well, if the paper by Simone Peters and colleagues [1] (open-access here) is to be believed the answer may very well be yes, at least in some cases, as they report a link between gluten consumption and feelings of depression under [short-term] experimental conditions. If replicated, such a finding may have profound consequences for how we view our relationship between food and mental health and wellbeing.
Bread Ma'am? @ Wikipedia 

I was initially drawn to the Peters paper as a function not only of the subject matter but also the authorship team. Mention of Jessica Biesiekierski in amongst the list of contributors immediately brought back memories of another ground-breaking paper of hers [2] reporting that "Non-celiac gluten intolerance may exist" (see here).

For those who might not know about non-coeliac gluten sensitivity (NCGS), this is a suggestion that outside of the classical connection between gluten and the autoimmune condition coeliac (celiac) disease, there exists something of a spectrum of gluten-related health issues. Some of these issues might have far-reaching implications for an array of conditions including that of autism... at least some autism (see here).

The Peters paper is open-access but a few details might be worthwhile mentioning:

  • This was a gold-standard trial in terms of being randomised, double-blind, placebo-controlled and including a cross-over component. What this means is that participants with irritable bowel syndrome (IBS) who were negative for coeliac disease (CD) (though not necessarily tested for the serology of CD) were "asked that they continue on a GFD [gluten-free diet] low in FODMAPS [Fermentable, Oligo-, Di-, Mono-saccharides And Polyols]" for the study duration. Participants were then divided up into groups to receive one of three dietary challenges to their gluten-free diet: gluten supplement (16g/day), whey supplement (16g/day) or placebo for 3 days followed by a wash-out period and then onto the next dietary supplement regime. Neither participants nor researchers knew who got what challenge when.
  • As well as adherence to the gluten-free diet and gastrointestinal (GI) symptoms being assessed, salivary cortisol secretions were measured alongside mental state as per the use of the State Trait Personality Inventory (STPI), a self-report measure.
  • Results: bearing in mind the relatively small participant group examined (n=22) and the various complications one might expect from undertaking a dietary trial, there was an "increase in [the] STPI state depression score following gluten ingestion compared to placebo". The depression scores were also higher when comparing gluten and whey supplements but did not reach statistical significance. 
  • No differences were found with regards to cortisol concentrations across the groups and time-frames nor for GI symptoms.
  • The authors conclude that: "Short-term exposure to gluten specifically induced current feelings of depression with no effect on other indices or on emotional disposition". They added: "Such findings might explain why patients with non-coeliac gluten sensitivity feel better on a gluten-free diet despite the continuation of gastrointestinal symptoms".

I have to say that as preliminary as these results might be, I'm quite excited at the implications from them. Yes, I have some professional interest in the relationship between gluten and our mental health / psychology and the so-called gut-brain axis (see here) so am perhaps biased, but to see some research utilising the gold-standard of evidence-based medicine coming up with the findings that they did is something rather grand. As per my opening sentences, replication, replication, replication is the next stop and then we can get really excited.

I note the authors do speculate on why they got the results they did. Cortisol is kinda ruled out as a cause as a function of their findings of no significant change. I might however draw your attention to other findings in this area which should be kept in mind. Serotonin (5-HT) also gets a mention as a potential explanatory reason, which is also interesting bearing in mind the mood connection with this neurotransmitter with appropriate caveats.

Then to some other interesting avenues for further study such as those trillions of beasties which call us and our gut home: the gut microbiota and their potential connection to our mood and psychology (see here). And how about a possible role for gluten exorphins and further the suggestion that opiate antagonists such as naltrexone might be a useful thing to look at? I'd perhaps also be minded to suggest that another possibility not discussed by the authors might be that of intestinal hyperpermeability (leaky gut) also playing a potential role given what we know about gut permeability and gluten [3]. More investigations like this please.


[1] Peters SL. et al. Randomised clinical trial: gluten may cause depression in subjects with non-coeliac gluten sensitivity – an exploratory randomised clinical study. Alimentary Pharmacology & Therapeutics. 2014. doi: 10.1111/apt.12730

[2] Biesiekierski JR. et al. Gluten causes gastrointestinal symptoms in subjects without celiac disease: a double-blind randomized placebo-controlled trial. Am J Gastroenterol. 2011 Mar;106(3):508-14.

[3] Vazquez-Roque MI. et al. A controlled trial of gluten-free diet in patients with irritable bowel syndrome-diarrhea: effects on bowel frequency and intestinal function. Gastroenterology. 2013 May;144(5):903-911.e3.

---------- Peters SL, Biesiekierski JR, Yelland GW, Muir JG, & Gibson PR (2014). Randomised clinical trial: gluten may cause depression in subjects with non-coeliac gluten sensitivity - an exploratory randomised clinical study. Alimentary pharmacology & therapeutics PMID: 24689456