Gut microbes and health

I'd like to apologise to my Gutness Gracious Me blog. I've neglected you and your subscribers for quite a few months now and I'm sorry. I have no excuse so please accept my humble apology.

Friends again?

OK. There's been quite a bit of new research come out in the intervening months which I need to catch up on. For this entry I'm going to list a couple of interesting papers showing how, if it was ever needed, gut bacteria really are starting to be taken quite seriously in lots of science circles.

Nature is a good starting point (the journal Nature not nature in general). A whole supplement was recently dedicated to gut bacteria which can be viewed here. Unfortunately the papers aren't open-access but I might draw your attention to two of them in particular:

• Valentina Tremaroli & Fredrik Bäckhed* discuss the "functional interactions between the gut microbiota and host metabolism" and how gut bacteria might, just might have the ability to affect how we derive energy from our food. I suppose this crosses quite a few different issues such as 'is a calorie a calorie' and how overweight and obesity might not just be a simple 'energy in - energy out' relationship. Indeed the concept of gut bacteria potentially regulating metabolism and absorption of things like fatty acids as per this paper by Semova and colleagues** (discussed here) may very well also be relevant.
• The paper by Craig Maynard and colleagues*** highlights another important function of our gut bacteria with regards to immune function and how the communication between the two systems may have the ability to influence our health and ill-health. Other authors have similarly speculated on this process as per this study by Cucchiara and colleagues**** using pediatric inflammatory bowel disease as an example.

Next up is another Nature journal, Nature Neuroscience, which carried an interesting piece by Cryan & Dinan***** on the potential 'mind-altering' effects of gut bacteria and everyone's favourite term: the gut-brain axis. I've kinda done bacteria potentially influencing behaviour before on a sister blog entry (see here) so don't really want to rehash that again. Suffice to say that as well as being home to quite a lot of bacteria, our gut also houses quite a few neurotransmitters and their receptors more traditionally associated with brain, so why would we expect these not to potentially serve functions other than controlling gut motility and the like. Whether there is interaction between these neurotransmitters and gut bacteria.... well I'd speculate there might very well be.

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* Tremaroli V. & Bäckhed F. Functional interactions between the gut microbiota and host metabolism. Nature. 2012; 489: 242-249.

** Semova I. et al. Microbiota regulate intestinal absorption and metabolism of fatty acids in the zebrafish. Cell Host & Microbe. 2012; 12: 277-288.

*** Maynard CL. et al. Reciprocal interactions of the intestinal microbiota and immune system. Nature. 489: 231-241.

**** Cucchiara S. et al. Interactions between intestinal microbiota and innate immune system in pediatric inflammatory bowel disease. Journal of Clinical Gastroenterology. 2012; 46: S64-S66.

***** Cryan JF. & Dinan TG. Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nature Reviews: Neuroscience. September 2012.

General Zod? No, General Zonulin

I hold my hands up and admit that gut hyperpermeability - leaky gut - is a bit of an obsession of mine. I know to some the mere mention of leaky gut conjures up images of 'alternative medicine' and all things tree-hugging. Just for the record I've never knowingly hugged any tree and am a meat eating, petrol car driving, house dwelling regular guy who has yet to dabble in anything 'alternative' assuming that the odd vitamin D tablet and probiotic counts as regular. Still I remain very interested in how abnormal gastrointestinal (GI) permeability might be tied into quite a few conditions.

One aspect of GI permeability in particular has surfaced quite recently on my research radar, an interesting compound called zonulin.

A description first. Zonulin enjoys quite a special place in the science of the tight junctions. Tight junctions (TJs) among other roles, serve quite an important barrier function in lots of parts of the body; so making sure that things stay in and other things stay out. Zonulin seems to be part and parcel of the chemistry of tight junctions and in particular sharing quite an important relationship with the enhanced permeability of TJs.

With the gut in mind, zonulin has found quite a bit of interest. A familiar name to this blog, Dr Alessio Fasano, seems to have been present right at the beginning of interest in zonulin, with a particular focus on gut permeability tied into the presence of coeliac (celiac) disease as per this article* and write-up.

Ever since then, zonulin has just been making wave** (full-text) after wave*** (full-text) after wave**** as per its 'disassembly' activity when it comes to TJs. The initial link with coeliac disease is an interesting one given that later work suggested that gluten, or rather the gliadin fraction of gluten, has the propensity to induce zonulin release***** (at least under certain laboratory conditions).

But coeliac disease was just the starting point for zonulin, as more recent research has suggested a potential role for this protein in relation to gut permeability in type-1 diabetes (here and here), obesity (here) and potentially quite a few other conditions (here) with a specific focus on autoimmune conditions. General Zod? No, General Zonulin.

Accepting that there still remains some work to do on zonulin with regards to the methods and mode of action of zonulin on gut permeability******* this is a very interesting protein.

With my 'wondering' hat on, I have a few questions:

• Assuming the link between type-1 diabetes, gut permeability and zonulin holds up, does this mean that a gluten-free diet might be 'advantageous' for at least some people with type-1 diabetes? I'm thinking about this recent case study******* as a template. I would also add that no medical advice is given or intended by  this question.
• Gut hyperpermeability, leaky gut, has been documented in other conditions including one close to my research heart, autism spectrum conditions (see here). Again noting the suggestions by de Magistris and colleagues (here) on how a gluten- & casein-free diet seemed to affect measures of gut permeability in their cohort, is it perhaps time to look at zonulin with regards to conditions like autism? How about schizophrenia also? 
• Finally(!), the amino acid glutamine and its proposed tie up with gut permeability. Might glutamine affect zonulin production or even the other way around? Or am I just confusing things and heading out a step too far? 

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* Fasano A. et al. Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease. Lancet. 2000; 355: 1518-1519.

** Wang W. et al. Human zonulin, a potential modulator of intestinal tight junctions. Journal of Cell Science. 2000; 113: 4435-4440.


*** Fasano A. Intestinal zonulin: open sesame! Gut. 2001; 49: 159-162.


**** El Asmar R. et al. Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure. Gastroenterology. 2002; 123: 1607-1615.


***** Clemente MG. et al. Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function. Gut. 2003; 52: 218-223.


****** Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Annals of the New York Academy of Sciences. 2012; 1258: 25-33.


******* Sildorf SM. et al. Remission without insulin therapy on gluten-free diet in a 6-year old boy with type 1 diabetes mellitus. BMJ Case Reports. June 2012

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The Human Microbiome Project

13th June 2012. The Human Microbiome Project reports first results on the bacterial constitution of 242 healthy adults sampled over 15-18 body parts up to three times. Framework results can be viewed here* (full-text) alongside what they found results here** and represent a bit of a milestone in our beginning to understanding how the trillions of bacteria which inhabit the human body, either in it or on it, play an important role in our lives.

I have to say that I am pretty excited about these papers and the other results published which can all be found at the PLoS Collections site (here). There is a massive of amount of material to go through which I want to talk about in future posts. For now, browse through a moment in scientific history.

* The Human Microbiome Project Consortium. A framework for human microbiome research. Nature. June 2012
DOI: 10.1038/nature11209

** The Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature. June 2012
DOI: 10.1038/nature11234

Coeliac disease as a model of autoimmunity

A very short post primarily to link to a recent paper by Kumar and colleagues* (full-text, at least for the moment).

The paper is a sort of 'all you ever wanted to know about coeliac / celiac disease but were afraid to ask' type piece and jolly informative in terms of what we think we know so far about the condition regarding genes, heritability, biochemistry and the like.

Just for good measure, here also are a few other links to some interesting discussions appearing in Nature recently on our gut microbiota and the Barker hypothesis. All I will say is 'all hail our gut bacteria' and onwards with the epigenetic revolution.

Happy reading!

P.S. I know I have been neglecting this blog for a few weeks and apologise. I will hopefully devote more time to it as the year goes on. In the meantime, a link to a peculiar song which has been running through my mind for a few week now.. Gotye and 'Somebody that I used to know' (UK readers might have heard this as an advert for 2Day 2012 from BBC Radio 2).

* Kumar V. et al. From genome-wide association studies to disease mechanisms: celiac disease as a model for autoimmune diseases. Seminars in Immunopathology. May 2012
DOI: 10.1007/s00281-012-0312-1

Gut microflora, coeliac disease and introducing gluten

A new paper by Sellitto and colleagues* (full-text) has been causing quite a bit of interest in certain circles. The paper as the name suggests is [partially] a 'proof of concept' study which includes several topics of interest for this blog with its focus on gut microflora (and dysbiosis), coeliac (celiac) disease (CD) and some interesting metabolomics science. The added value comes with the name Alessio Fasano as part of the authorship list.

There is quite a bit of details to this paper but in essence the aims were: (i) to characterise the changes from birth to 24 months in terms of gut bacteria to genetically at-risk of coeliac disease children as a function of early or delayed introduction of gluten to the diet, and (ii) to undertake a range of analytical methods to map such bacterial populations with the hope of further informing on any relationship between gut bacteria and coeliac disease.

The paper is full-text but a quick summary of proceedings and findings:

• Forty-seven infants who had one parent with biopsy-proven CD were initially recruited before weaning had commenced. All were breastfed from birth to at least 6 months of age. From 6-12 months of age, 30 infants positive for either the HLA DQ2 and/or HLA DQ8 genotypes were randomly allocated to either a gluten-free - delayed gluten exposure - group (n=13) or a gluten load - early exposure - group (n=17).
• A smaller number of children from each group (n=8 each) were selected randomly for the analytical side of the study (which is what this paper in essence reports) where stool samples were collected at various points over the study period ranging from 7 days in to 24 months.
• The results: none of the 8 infants from the delayed gluten exposure group developed CD over the course of the study. One of the 8 infants in the early gluten introduction group did go on to develop CD at 2 years of age as measured by various serological panels and went on to a gluten-free diet with a remission of serology at follow-up.
• When levels of anti-gliadin antibodies (AGA) (IgG) were examined and corrected for gluten exposure time, the early gluten introduction group showed a higher number of IgG-AGA positive results than the delayed exposure group. The authors discuss how AGA is not necessarily a particularly good measure of CD but could indicate greater levels of intestinal hyperpermeability (leaky gut) as a result of exposure to the gliadin fraction of the gluten protein in the same way that IgG levels have been interpreted in other studies (see Sutterella and autism post). 
• Pyrosequencing of the various species and families of bacteria present across the groups at different time frames suggested some interesting goings-on. To quote: "the GI tract microbiota in DQ2+/DQ8+ infants appears to be lacking significant numbers of member of the phylum Bacteroidetes". That and a higher abundance of Firmicutes, implies that maturationally, the gut microflora of children at elevated risk of CD is different from lower risk groups as determined by comparison with an external dataset** (full-text).
• The application of 1H-Nuclear Magnetic Resonance Spectroscopy (NMR) to proceedings added that metabolomic touch as "..SCFA succinate, acetate, propionate and butyrate are found in the feces" following the introduction of solid foods. Having said that little distinguishing data was found to categorise the two groups in any significant, universal way.

It's taken me a while to get my head around all the findings from this recent paper because there was a lot of data produced bearing in mind the small participant group and preliminary status of the paper. One of the first things that did strike me is the overlap in these findings and some fairly recent data published looking at carbohydrate metabolism and autism included in this post. Decreasing Bacteriodetes and increasing Firmicutes was the preliminary finding from Williams and colleagues*** bearing in mind the difference in samples being analysed and the lack of data on HLA DQ2 / HLA DQ8 genotype provided in the group with autism being studied. I'm not going to say too much more of this 'similarity' aside from the fact that screening for coeliac disease and/or excessive intestinal permeability perhaps ought to be much more commonly undertaken in cases of autism spectrum conditions just to rule them out.

Quite a few mentions of the word 'dysbiosis' are also recorded in the current paper which reaffirms the possibility of a connection between the various populations of bacteria that reside within us and our potential risk of disease. I like the idea that this study looked at both the metabolomic and genomic side of things even if it was just based on the HLA DQ geneotypes.

So from the starting point of a genetic susceptibility to gluten, we have preliminary data on functional changes to the microbiome in susceptible people and some interesting tools for looking at how this might be expressed functionally. I look forward to more studies of this type with greater participant numbers, and in particular how such findings might extend into other autoimmune conditions and even beyond just somatic presentation.

* Sellitto M. et al. Proof of concept of microbiome-metabolome analysis and delayed gluten exposure on celiac disease autoimmunity in genetically at-risk infants. PLoS ONE. March 2012
DOI: 10.1371/journal.pone.0033387

** Palmer C. et al. Development of the human infant intestinal microbiota. PLoS Biology. 2007; 5:e177
DOI: 10.1371/journal.pbio.0050177

*** Williams B. et al. Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances. PLoS ONE. September 2011.
DOI: 10.1371/journal.pone.0024585

Strong intestinal barrier and the big C?

I enjoy reading the odd newspaper now and again just to find out what's going on in the world. Having said that I do take some of the headlines with a pinch of salt as evidenced by a recent UK headline about autism which has been roundly brought up on its inaccuracy.

With such things in mind, I approach this post with very much more caution than usual given the subject matter - cancer - and the propensity for such headlines to become 'over inflated'. The headline in question came from this news piece on the recent publication by Lin and colleagues* (open-access) on a possible connection between the compound guanylyl cyclase C (GC-C) and the integrity of the intestinal barrier which might have onward repercussions outside of just malabsorption issues.

Let's start from the beginning on this one. Guanylyl cyclase C (GC-C) is, as its -ase name suggests, an enzyme found in gut and brain. It plays a role in regulating intestinal fluid and balancing electolytes. For those brave souls who quite like a bit of heavy biochemsitry, quite a thorough description of the whole guanyly cyclase family can be found here. Going back to GC-C, more recently, evidence has been accumulating to suggest that GC-C might also have some connection to intestinal barrier function** based on knockout mice studies.

The recent study by Lin went one stage further suggesting that in a mouse model GC-C did indeed link to barrier integrity through its effect on various junction proteins. It also however affected oxidative DNA damage when silenced subsequently ".. associated with increased spontaneous and carcinogen-induced systemic tumorigenesis".

Some details:

• Various mouse models were used; the important ones being mice deficient in GC-C (called GUCA2A in this paper) which will be called GC-C-/- (the -/- denoting zygosity for the receptor, as in homozygous for deficiency) and mice GC-C+/+ (denoting homozygous for no deficiency).
• A few differences came to light between the -/- and +/+ mice: the -/- mice produced less tight junction proteins including occluden, claudin-2, claudin-4 and JAM-A. In English, these are some of the main constituents that keep the gut barrier in good integral health. This was confirmed when looking at intestinal permeability which was increased (more leaky) in the -/- mice.
• When trying to chemically induce intestinal barrier issues via DSS, the severity of the colitis produced was increased in the -/- mice compared with the +/+ mice; something also seen in the mortality-survival rates between the two models.
• A quote from the paper: "Impaired basal epithelial barrier integrity producing systemic genotoxicity was associated with spontaneous extra-intestinal tumorigenesis, including tumors in mesenteric lymph nodes, livers, and lungs, in 50% of Gucy2c−/− mice, but in only 10% of Gucy2c+/+ mice". Translation: more permeability in the gut of the -/- mice led to more tumours in other organs compared to +/+ mice.

There is a lot more to this paper which I unable to cover in this short post. The one obvious point to make is that this was a mouse model of GC-C deficiency and hence needs a little more investigation into whether such processes transpose so readily on to humans. GC-C already has a possible link to metastatic cancer cells as per articles like this one so one would already expect quite a bit of interest in this compound in cancer research circles.

Combined with my previous post on diabetes and leaky gut, it is heartening to see some novel research is being done on how gut barrier permeability might not necessarily just manifest in intestinal symptoms. I leave you with another quote from one of the authors: ".. if you want to prevent inflammation or cancer in humans, then we need to start thinking about feeding people hormones that activate GC-C to tighten up the [intestinal] barrier.” I am certainly not advocating this or any other 'advice' at this time but perhaps this is fodder for further research and a later post methinks.

* Lin JE. et al. GUCY2C opposes systemic genotoxic tumorigenesis by regulating AKT-dependent intestinal barrier integrity. PLoS ONE. February 2012.
DOI: 10.1371/journal.pone.0031686

** Han X. et al. Loss of guanylyl cyclase C (GCC) signaling leads to dysfunctional intestinal barrier. PLoS ONE. 6: e16139
DOI: 10.1371/journal.pone.0016139

Does diabetes start in the intestines?

The findings of an interesting paper by Wei and colleagues* (full-text) pose a question: are the origins of diabetes in the intestines?

For those that don't know too much about diabetes, here is a link that should help. The concise version (if there is such a thing) is that diabetes normally manifests as either type-1 diabetes or type-2 diabetes with insulin being the key compound in controlling blood sugar, and corresponding issues either with its production or when resistance is built up to it.

The crux of the paper by Wei et al is that an insulin-responsive super enzyme called fatty acid synthase (FAS) involved in lipogenesis is also involved in gut barrier regulation through its action on Mucin 2 (Muc2), a gel-forming component of mucus. The authors' suggestion is that becoming resistant to insulin is associated with issues with FAS and correspondingly problems with mucus in the gut, inflammation and diabetes. No pressure then.

The paper summarised (deep breath):

• Several groups of mice were included for study: (a) mice with chemically-induced (tamoxifen induction of Cre recombinase) decreases of FAS protein and mRNA, (b) mice bred with inactivated FAS in the intestine and (c) control germ-free mice. For group (b) mice, diabetes was induced by administration of streptozotocin, a toxin to the beta cells which produce insulin in the pancreas.
• Assays looking at gut bacteria, intestinal permeability, cytokine release and protein S-palmitoylation were used to investigate various parameters.
• The findings: a chemically-induced deficiency of FAS in mice started a cascade of events linked to inflammation. One of the primary cytokine markers of this inflammation was elevated levels of TNF-α although animals were also noted to show weight loss and other gastrointestinal symptoms. A quarter of these mice actually died within 14 days.
• The authors deduced that although some changes were noted to the intestinal bacterial makeup of FAS reduced mice, these changes were not enough to cause the inflammation observed but rather were as a result of the inflammation. They demonstrated this via a previously discussed method on this blog, bacterial transplantation; in this case to the germ-free mice (group c) who did not show the accompanying inflammation as a result of their donor bacteria. That is not however to say that gut microbiota did not have some effect, as per the reduction in inflammation noted in the FAS deficient mice following administration of the antibiotics ciprofloxacin and metronidazole.
• The link between FAS deficiency and Muc2 was evidenced by the lower levels of Muc2 shown in FAS deficient mice and reduced inner mucus layer thickness in the colon of affected mice. 
• Looking at the inactivated FAS (group b) diabetic mice, a similar pattern of issues with Muc2 and reductions in the mucus layer was seen alongside penetration of bacteria indicating intestinal hyperpermeability (leaky gut). Interestingly, insulin supplementation seemed to positively affect some of the permeability issues.

This is quite a complicated paper and so please do not take my summary as gospel. It is intriguing that inflammation is at the heart of their theory and in particular, inflammation as a result of not having enough FAS present in the gut with the knock-on effects on gut permeability. Indeed not for the first time has it been suggested that diabetes and leaky gut are connected as per articles like this one. Makes you wonder also about any other possible dietary inter-related connections?

* Wei X. et al. Fatty acid synthase modulates intestinal barrier function through palmitoylation of mucin. Cell Host & Microbe. February 2012.
DOI:  10.1016/j.chom.2011.12.00

On gut parasites and chronic fatigue

An interesting exchange on Twitter prompted this short post regarding a paper by Naess and colleagues* (full-text) on Giardia lamblia gastroenteritis and chronic fatigue syndrome (CFS). The tweets concerned another parasitic nasty called Toxomplasma gondii which has featured quite a bit on a sister blog with regards to its link to various behaviourally-defined conditions. I thought that T.gondii was a spine-tingling protozoa until someone posted about these other chaps and their brain-eating, behaviour-changing and belly exploding antics (pass the sauce, please).

Giardia lamblia is quite a special  protozoa in terms of its survival, persistence and ability to link into quite a few other health complaints particularly of the gastrointestinal variety and specifically links to lactose intolerance. The current observations by Naess et al are interesting in that based on an examination of over 1200 patients with laboratory-confirmed giardiasis following a large community outbreak in Bergen, Norway, approximately 5% of cases (58/1262) were diagnosed with CFS as classified by the CDC criteria (see here for a related post on the trials and tribulations of diagnosing CFS/ME).

Even assuming a CFS prevalence of 1% previously noted in children (not adults) in the UK, the 5% figure seems high bearing in mind correlation is not necessarily causation. What can perhaps be ascertained from this latest study is that it might be a good idea to screen for giardiasis where active functional bowel issues are present alongside fatigue-related conditions and further research on any mechanism of parasitic infection linked to long-term fatigue might be advisable.

* Naess H. et al. Chronic fatigue syndrome after Giardia enteritis: clinical characteristics, disability and long-term sickness absence. BMC Gastroenterology. February 2012.
DOI: 10.1186/1471-230X-12-13

Plasma amino acids and inflammatory bowel disease

My recent post on the application of metabolomics to food fingerprinting got me thinking about the examination of biological fluids and how our metabolome might have some interesting secrets to one day share. The 'promise' of the science of metabolomics is that one day, we should be able to look at various biological fluids across a range of conditions and based on the compounds excreted/detected determine diagnosis, disease progression and how well someone responds to intervention. I hasten to add that we are nowhere near that position yet.

In light of this, it was perhaps inevitable ('it is your destiny') that this paper by Hisamatsu and colleagues* (full-text) on possible plasma biomarkers for inflammatory bowel disease (IBD) would get some attention. I'm pretty sure that I don't have to explain this to viewers but the term inflammatory bowel disease covers quite a bit of diagnostic ground including Crohn's disease and ulcerative colitis. Diagnostic confirmation of these conditions is quite a complicated process normally involving a combination of peripheral measures (blood tests, stool analysis) coupled with more direct observation of the bowel.

Hisamatsu and colleagues reported that a previously trialed network analysis of plasma amino acid levels in patients diagnosed with IBD might provide novel, non-invasive and importantly, objective biomarkers potentially opening up some new research areas into the nature of IBDs.

The details:

• Fasting plasma 'aminograms' for a discovery group of 102 Japanese adult patients diagnosed with Crohn's disease (CD) and 102 patients with ulcerative colitis (UC) were initially compared against 102 healthy control participants. The majority of participants were male (70%) with disease duration ranging between an average of 7.8-11 years. A minority of participants were described as having 'active' disease (CD = 29/102; UC = 38/102).  Obtained aminogram results were also validated using a validation set of participants (CD: n=63; UC: n=120; controls: n=108).
• Serum albumin levels were reported as significantly lower in the IBD groups compared to controls.
• Various differences were noted between the groups in terms of amino acid levels. The authors seemed to have concentrated on histidine and tryptophan as primary examples with a view to disease activity and correlation with C-reactive protein levels (a marker for inflammation).
• Implementing their network analysis (MIAI), the authors came up with a formula based on 6 amino acids which discriminated CD and UC groups from controls with ROC values ranging from 0.894 to 0.955 depending on whether the discovery or validation group were used and comparing across the IBDs with controls. 
• Depending on whether disease was active or in remission, a formula incorporating data for 7 amino acids was suggested to have some power in discriminating disease activity with ROC values ranging from 0.894 (CD active vs. CD remission) and 0.849 (UC active vs. UC remission).

The data produced by this study is interesting. OK, it is not totally 'diagnostic' either in terms of classifying IBD from controls or looking at active vs. remissive symptom presentation but it's not a bad start at all. Indeed the use of discovery and training sets takes me back to a wonderful paper covered last year on schizophrenia, which did find a perfect classification based on a handful of serum and urinary markers. It makes me wonder if they were looking at serum and urine at the same time or maybe incorporating a few more well-known markers, whether those ROC values might further approach the magical number 1 (denoting a perfect classification).

The use of statistical models allied to biochemical data is also interesting. I note that similar linkages have been used in other areas of medicine, possibly even to recreate speech from brain activity...?

* Hisamatsu T. et al. 2012 Novel, objective, multivariate biomarkers composed of plasma amino acid profiles for the diagnosis and assessment of inflammatory bowel disease. PLoS ONE. January 2012
DOI: 10.1371/journal.pone.0031131

Newsflash: defining gluten-related disorders

In the style of one Homer J Simpson... can't stop... must finish for the day.. new guidance of what constitutes a gluten-related disorder just published by Sapone and colleagues*.

If there is one document that you absolutely have to look at which summarises where we are in relation to gluten-related conditions, not just coeliac disease, this is it.

It's full-text, has a myriad of gluten research names included on it (including Alessio Fasano, Marios Hadjivassiliou and David Sanders), so enjoy.

* Sapone A. et al. Spectrum of gluten-related disorders: consensus on new nomenclature and classification. BMC Medicine. February 2012.
DOI: 10.1186/1741-7015-10-13

Pesticides and vitamin D deficiency

Chemistry World, the public face of the UK Royal Society of Chemistry (RSC), carried an interesting report recently discussing research linking exposure to organochlorine pesticides (OCs) with vitamin D deficiency. The research in question is this paper by Jin-Hoon Yang and colleagues* (full-text) and before you ask, yes, it is a study of 'association', so we tread carefully.

Before wading into this study it is interesting to note that vitamin D is currently enjoying quite a trendy following in many areas. Important discussions are underway to determine whether a resurgence of conditions like childhood rickets means supplementation needs to be more closely inspected. 

I digress. A summary of the paper in question:

• An analysis of plasma levels of 7 OCs (including DDE and DDT) was undertaken for 2,337 people aged 12 and above recruited as part of the US National Health and Nutrition Examination Survey (NHANES). After exclusion of those where accompanying 25-hydroxyvitamin D (25(OH)D) were not available alongside other exclusions (e.g. pregnant women, being under 20 years old), the final participant group number was N=1275.
• The results: based across various grouping on concentrations of OCs, there were quite a few significant inverse relationships reported. That is, for the OCs -  p,p′-DDT, p,p′-DDE, and β-hexachlorocyclohexane - elevated plasma levels of these compounds individually and collectively were associated with lower vitamin D levels. Plasma levels of DDT in particular showed quite an enduring association with vitamin D levels. 
• There is a suggestion that the relationship may also be dose-dependent up to a certain point; that is up to a value of 200 ng/g lipid of DDT, vitamin D levels dropped, but increasing levels of DDT after that seemed to be linked with increasing vitamin D levels bearing in mind that most participants presented below this 200 ng/g lipid threshold.

As I said, this is a study based on association. Controlling for factors such as gender, age, race and vitamin D supplementation is an admirable quality of the study but association is normally only a guidepost to a possible relationship, not a dead cert. That and the fact that little information has been provided on why the results came out as they did, leaves the door open to further study in this area.

Noting that this blog is primarily concerned with the gut, I do wonder about these recent findings and how they may (or may not) fit into a past post on gut bacteria and organochlorine pesticides. It's a bit of a long shot but how about testing the suggestion that OCs affect gut bacteria which in turn affects other systems including vitamin D and its receptors?

* Yang J-H. et al. Associations between organochlorine pesticides and vitamin D deficiency in the U.S. population. PLoS ONE. January 2012. DOI:10.1371/journal.pone.0030093

Metabolomics and the food fingerprint

I've touched upon the beautiful science of metabolomics previously on a sister blog; that is, looking for 'chemical fingerprints' in various biological fluids to garner further information on underlying biological processes related to health. Already metabolomics has provided us with a few preliminary clues in relation to more behaviourally-led conditions such as autism and schizophrenia (AUC=1), but as the technology gets smarter and smaller (and cheaper), I foresee great things ahead for this flourishing branch of biochemistry similar to the genomic high-throughput screening technology carried out at places like the Sanger Institute.

When a story recently appeared on the BBC website discussing a food 'fingerprints' test, it was therefore always going to attract my attention. Indeed so much so that after a few quick emails to one of the people involved with the research, I was very satisfied to receive some of their peer-reviewed papers on the details behind the headlines. 

The BBC story discusses some collaborative research based on the notion that what we eat leads to the production of various chemical metabolites and that detecting those metabolites in fluids like urine means we can objectively find out what someone has consumed recently and if necessary promote any dietary changes. Food diaries are the normal way of collecting information on what people have eaten recently but recall is often not as accurate as you think.

My take on this work was slightly different in terms of the fact that the researchers are in effect, building up a large bank of information based on very controlled eating habits about what comes out when we eat certain foods. 

This paper by Favé and colleagues* (full-text) sums up the research aims and objectives; also providing a great overview of the various techniques used in metabolomics and importantly the statistical analyses required (normally PCA or some variant). The researchers have been very methodical in their approach to this issue. This paper again by Favé and colleagues** (full-text) details how they went about clearing a few potential interfering variables such as the fasting period and some information on the reproducibility of findings over various testing occasions. In short, they wanted to make sure that interesting collected metabolites were food derived and were consistent.

So what have they got so far?

Well, according to this paper by Lloyd and colleagues*** when compared to a standard breakfast made up of orange juice, tea with skimmed milk and sugar, butter croissant, and cornflakes with milk, an alternative dietary schedule made up of either salmon, broccoli, whole-grain wheat cereal or raspberries produced some notable differences in urinary metabolites. The differences were even more notable when the alternative dietary schedule was compared against fasting urine samples.

Looking more specifically at individual compounds formed as a result of dietary intake, a few interesting snippets were reported including:

• Smoked salmon intake was associated with increased levels of 1-methylhistidine and anserine (b-alanyl-L-methylhistidine), metabolites of the amino acid histidine, probably derived from fish skeletal muscle. Also alongside were findings of TMAO, a degradation product from carnitine (remember that?).
• Broccoli and raspberry were both associated with increased ascorbate and individually, xylonate/lyxonate and polyphenols.
• For each of these three dietary components, there were at least 7 putative biomarkers of intake reported although nothing was reported on for the wholegrain cereal consumption.

Another paper by Lloyd and colleagues**** reported that proline betaine was associated with citrus exposure, with detected levels using the same technology and methods explanatory of both acute and habitual exposure to citrus containing foods (makes it sound like an illicit drug or something).

Appreciating that this and related work is still an emerging area of investigation, subject to all manner of confounders that we as human beings do to ourselves every day, I am suitably impressed that there are groups out there looking at our food and how we metabolise it. I can envisage many applications for this kind of work, ranging from a non-invasive test of recent food consumption, to measuring adherence to certain kinds of dietary intervention, to examining whether there are any differences in the metabolism of certain foods as a function of say gut bacteria which might cast some light on the biology behind lots of conditions.

To finish how about a bit of artistic interpretation through the medium of dance from Kate Bush...

* Favé G. et al. Measurement of dietary exposure: a challenging problem which may be overcome thanks to metabolomics? Genes & Nutrition. 2009; 4: 135-141

** Favé G. et al. Development and validation of a standardized protocol to monitor human dietary exposure by metabolite fingerprinting of urine samples. Metabolomics. 2011; 7: 469-484

*** Lloyd AJ. et al. Use of mass spectrometry fingerprinting to identify urinary metabolites after consumption of specific foods. American Journal of Clinical Nutrition. 2011; 94: 981-91.

**** Lloyd AJ. et al. Proline betaine and its biotransformation products in fasting urine samples are potential biomarkers of habitual citrus fruit consumption. British Journal of Nutrition. 2011; 106: 812-824.

Greens, glutathione and the mucosal barrier

'Eat your greens' is a phrase most of us will have heard on several occasions when growing up. I have to admit to being a bit of a 'goody two-shoes' in this respect, having had a lasting affection for things like Brussels sprouts and broccoli. Indeed, sprouts still tend to be on my menu several times a week (boiled with a drop of Greek olive oil).

I say all this because an interesting article published a few years back has caught my eye recently. The paper by Hoensch and colleagues* (open-access) looked at the various factors potentially affecting the functioning of the gastrointestinal glutathione system with some interesting observations.

I will admit that glutathione (GSH) is something which I am becoming very interested in at the moment. Over on my autism research blog, I recently discussed the pretty remarkable findings emerging in some cases of autism with regards to plasma GSH which might (might!) eventually have some diagnostic usefulness in combination with other factors. The Hoensch paper talks about GSH levels in the upper gastrointestinal mucosa and how factors such as diet, some medications and even gender seemed to affect levels of GSH and accompanying enzyme activity (glutathione S-transferase, GST).

The particulars:

• Biopsy pinches taken from two sites (antral and duodenal mucosa) for 202 adults (104 males: 98 female) undergoing endoscopy were analysed for GSH and GST activities. Various background information was also taken from participants including details of family history, medication and current dietary habits via a food frequency questionnaire.
• The findings: different biopsy sites reflected different levels of GSH and enzyme activity. Female participants showed higher levels of GSH and GST activity in their antral mucosa samples than males. High intake of vegetables (more than 3 days a week) seemed to enhance aspects of GST activity.
• A subsequent author reply to a suggestion that Helicobacter pylori infection might also have had an effect suggested that indeed, one aspect of GST activity was negatively affected by H.pylori infection.

Bearing in mind that GSH and its related sub-systems represent an important part of our defences against those dastardly free radicals among other things, making sure that the system is in tip-top condition is probably quite important. I don't want to make sweeping gender generalisations but the fact that females seemed to be in a slightly more advantageous position compared with male participants leads me back to some interesting work on the fragile male. Having said that, let's not be too defeatist here; greater vegetable consumption seemed to have an enhancing effect as per other results so one could argue, guys in particular, eat more greens - especially more brassica vegetables to support your mucosal antioxidant defence system.

* Hoensch H. et al. Influence of clinical factors, diet, and drugs on the human upper gastrointestinal glutathione system. Gut. 2002; 50: 235-240.

Gut bacteria and heart health?

Happy (belated) New Year! Welcome back to Gutness Gracious Me in 2012. I start this year with a post on something pretty central to the ethos of this blog: gut bacteria.

I should perhaps first apologise to Alex Gazzola (#HealthJourno) who very kindly asked if I would be discussing the new Codex standards for labelling foods and gluten-free and the 20 parts per million threshold introduced. I did say I would have a look at this and I am still looking. I do however think that Alex has done a wonderful job of covering this topic on his own blog (see here) as per the reader response he has received.

Back to task. Despite being only 13 days into the New Year (Friday 13th...mmm?), already the research is coming thick and fast. Over at my Questioning Answers blog, the big news recently has been on the bacteria Sutterella and its detection via various methods in quite a few biopsy samples from children with autism and gastrointestinal (GI) issues. I say big news but in among the very detailed explanation of how that team came to find Sutterella, the main story for me is the suggestion that gut hyperpermeability in some cases of autism might be a route through which the immune system meets gut bacteria in places it really shouldn't and onwards formulates an antibody response. The template for this is Crohn's disease and leads into some interesting suggestions about whether gut bacteria itself might not necessarily be the bad guy but rather what happens when it is allowed to roam.

The other quite interesting news is from this paper by Vy Lam and colleagues* suggesting that different amounts and varieties of gut bacteria might influence the severity of and recovery from heart attacks in rats. I am well used to seeing papers talk about gut bacteria in relation to GI issues and conditions, even conditions like autism where functional and more systemic bowel disorders have been noted in some cases. This is however one of the first times that I have come across gut bacteria potentially so directly influencing the physical health of an organ like the heart.

So what did the researchers do?

• Three groups of rats were fed three different types of diet: a standard diet, a standard diet plus quite a powerful antimicrobial (vancomycin) and a standard diet plus a probiotic very aptly named GoodBelly. 
• The primary probiotic constituent of GoodBelly is Lactobacillus plantarum 299v quite commonly found in fermented foods like sauerkraut and subject to a few claims. One of those claims is that the bacteria might be able to reduce the production of leptin among other things.
• Both the vancomycin and GoodBelly supplemented groups showed a decrease in circulating leptin levels (38% and 41% respectively) alongside some changes to gut bacteria as one might expect.
• The vancomycin and GoodBelly supplemented groups also showed evidence of smaller heart attacks and improved recovery after heart attack compared with the standard diet group.

Whilst the authors describe this as a 'proof-of-concept' study, there are potentially many implications from this work not least that our gut bacteria might do far more than call us home and help digest our food. I have previously (jokingly) referred to gut bacteria as being our 'masters'. Assuming the results from this study are pertinent to humans as well as rats, I might well be offering a sacrifice to 'those who must be obeyed' in the near future.

Should we be surprised that the human body is interconnected? No probably not, although it might take a while and a few more bits of evidence of effect before cardio health care professionals start giving probiotics to help treat (prevent?) myocardial infarction (please note I am not giving any medical advice about this). Going back to the Sutterella post and the proposed link between bacterial translocation and gut permeability, I do wonder whether research should also be looking more closely at gut permeability and lots of other conditions just to see if findings such as these might provide a few more clues about health and illness.

*Lam V. et al. Intestinal microbiota determine severity of myocardial infarction in rats. The FASEB Journal. January 2012