- Structure and function of the gut
- Zonulin and tight junctions
- Zonulin in health and disease
- Factors that increase zonulin
- Are there any SNPs that alter zonulin activity or expression?
- How to check your zonulin levels
- Take-home message
What is zonulin and how does it play a role in the explosion of autoimmune conditions we are seeing in the west?
If you do a lot of reading about nutrition and genetic health you’ve likely come across the term “leaky gut”, or more technically increased membrane permeability, which some claim is the cause of a wide range of longterm symptoms such as chronic fatigue syndrome and multiple sclerosis, even ranging as far as autism and migraines. However, as far as the science goes these associations are currently unsupported, with the quote below from a recent review giving the most concise statement:
Although several aspects of barrier function can be assessed in man, one must be aware of exactly what a given test measures, as well as of its limitations. The temptation to employ results from a test of paracellular flux to imply a role for barrier dysfunction in disorders thought to be based on bacterial or macromolecular translocation must be resisted. Although changes in barrier function have been described in several gastrointestinal disorders, their primacy remains to be defined. At present, few studies support efficacy for an intervention that improves barrier function in altering the natural history of a disease process.
Quigley EM, Curr Opin Gastroenterol. 20162
I’ve bolded what I would consider to be the key statement. So whilst the evidence for a “leaky gut” as a driver of certain diseases is weak, there are numerous disorders which can both drive and result from its occurrence.
Before we get into the material on the structure and function of the gut, we have two recent podcast episodes that touch on these issues if you really want to go down the rabbit hole:
- The future of food sensitivity testing, curing IBS and rethinking gluten free diets
- The histamine leaky gut connection
Both of these episodes discuss zonulin and leaky gut in great detail.
Structure and function of the gut
Before we go on, let’s have a look at how the lining of the gut functions and how it could become leaky.
The inside of your gut is lined with a single layer of epithelial cells which make up the mucosal barrier. These cells are highly specialized having to allow absorption of all nutrients the body requires, whilst also stopping harmful compounds or invading microorganisms from passing across the mucosal barrier into the blood stream.
To do this, epithelial cells have two major protective features; firstly, they secrete mucus into the gut which acts to trap harmful microorganisms before they reach the epithelium. Secondly, they form an impermeable wall by linking together with so called “tight-junction” proteins. Importantly, as well as protective functions, these two features also promote healthy absorption of nutrients from the gut.
Image from Wikipedia
The tight junction proteins create a barrier between the inside of the gut (lumen) and the rest of the body to prevent harmful microorganisms passing into the blood, and also aid in the efficient uptake of nutrients.
So you can see that alterations in the mucus layer, or the formation of the impermeable barrier (going from “tight” to “leaky”) could have large health impacts limiting proper absorption of nutrients and allowing infection of the blood and other organs, a lot of which John covers in his excellent post covering candida.
Zonulin and tight junctions
Zonulin was discovered in 2000 by researchers who were investigating the action of Vibrio cholerae (the bacteria which causes cholera) and its secreted zonula occludens toxin (ZOT).3 ZOT is one of the toxins released by Vibrio cholerae which cause the severe diarrhoea experienced by those with cholera, and acts by loosening the tight junctions of the gut. As these weaken, water can rapidly flow back into the gut putting sufferers at severe risk of dehydration.
When studying this toxin, researchers identified that there was a human analogue which our own gut cells could release in order to regulate tight junction structure and function, which they named zonulin.4 Importantly, zonulin remains the only modulator of intracellular tight junctions expressed so far that can affect gut function and health and associated immune response and so it is widely investigated.
Zonulin in health and disease
So the first question researchers asked is, what diseases are associated with changes in zonulin activity or function?
As I discussed previously celiac disease describes a relatively common (it affects approximately 1 in 135 of us, although complications with diagnosis may mask the true number5), lifelong disorder of the digestive system, where the intestine becomes inflamed and is unable to absorb nutrients properly, or process our food correctly.5 You can see the effects on the lining of the gut below, the delicate villi and micro-villi are lost as the tissue has become inflamed.
The villi seen in healthy gut are almost completely lost in celiac patients, greatly reducing the surface area and impairing nutrient absorption.
If it we look at another microscope picture of the gut from a healthy individual and one with celiac, this time stained for claudin (red) and ZO-1 (green) you can clearly see how the staining is much less intense and covers a far smaller area, suggesting breaks have appeared in the intestinal lining.
Image from Schumann M, et al. Gut. 20116
Looking at specific tight junction proteins claudin (red) and ZO-1 (green) the integrity of the gut epithelium is clearly compromised with fewer tight junction proteins present.
Serum zonulin has been shown to be significantly up-regulated in people with celiac disease compared to healthy control7, and also in the cells of the gut themselves when they were assessed after biopsy.8
Type 1 diabetes
Type 1 diabetes (T1D) occurs when immune cells in the body attack and destroy beta cells in the pancreas leading to issues with insulin production and ultimately blood glucose control. Whilst some genetic markers for T1D have been described, the the causes of the remaining cases remains unknown.
The above image shows the pathway to the development of T1D.
A common feature of T1D are alterations in to the structure of the gut promoting a loss of tight junctions. These were typically described as symptoms of the disease9 (stage 3 or beyond in the above figure), however more recent studies have shown that loss of tight junctions actually occurs before disease onset (stage 1 or before in the above figure).10
Several studies subsequently indirectly (hence the slightly lower science grade) linked increased zonulin with the onset of T1D following exposure to dietary triggers such as gliadin and glutenin the two main components of gluten from wheat.1213
In both inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) changes to intestinal permeability have been observed in sufferers.1415 For IBS only a single study has been performed to investigate zonulin levels which reported no association 16, to date no study has been performed in people with IBD assessing zonulin although there are some promising findings based on mouse models.17
Multiple sclerosis is a disease which is characterised by an increase in blood-brain barrier permeability18 with more recent studies showing an increase in gut permeability as well.19 This same study also demonstrated that zonulin levels were twice as high in MS patients compared to healthy controls.19 However, this work has not been replicated and the chicken or egg questions remains to be answered; i.e. do changes to gut permeability lead to the development of MS, or are these changes an additional symptom of MS? Hence the currently low science grade rating.
Finally onto perhaps the most acute of the disorders discussed sepsis (sometimes called septicemia or blood poisoning). Sepsis is a serious (and thankfully relatively rare, although its incidence is increasing) condition whereby the body’s response to infection damages its own tissues and organs which if untreated can rapidly lead to organ failure and death.20
Numerous studies have reported increases in gut permeability associated with the onset of sepsis,2122 with a follow-up study demonstrating that zonulin was elevated in patients with sepsis.23 However, this study was rather small and the authors suggest that a clinical trial with more patients is required before definite conclusions are made.
Sepsis is of growing importance due to the developing issue of antibiotic resistance. Although an acute disorder if caught in time sepsis could be treated efficiently, however if a fully resistant bacteria was involved then our treatment options are significantly reduced.
Factors that increase zonulin
There are two main factors that have been identified to increase zonulin expression in the gut.
It is now well established that the bacteria which colonise our gut can have a large impact on our health, both positive and negative. Several studies have shown that certain bacterial species can induce the expression of zonulin.24 In turn this increased expression of zonulin is able to loosen tight junctions and increase gut permeability.24 What is unclear is if this is a normal physiological response by the body, trying to clear infection by flushing bacteria out of the gut, which may go awry leading to bacteria passing through the gut membrane. Or if it is a process driven by bacteria as a means to invade the body. Understanding this aspect will be key in the development of future therapies relating to zonulin.
The second main factor is gluten or more specifically two of its components gliadin and glutenin, both of which promote the release of zonulin.24 Interestingly researchers have shown that the cells of the gut only respond to gluten when it is present on their luminal (food side as opposed to body side) side. This allowed them identify a specific cell receptor (CXCR3) for gluten which was then shown to be over expressed in celiac patients.24
Are there any SNPs that alter zonulin activity or expression?
The zonulin protein is encoded for by the HP gene (zonulin is also sometimes called haptoglobin). Two allele variants of HP exist termed Hp1 and Hp2 and their distribution varies (Hp1-1 ~ 20%, Hp2-1 ~ 50% and Hp2-2 ~ 30%) amongst western populations.25 The Hp2 variant has been linked with a higher risk of developing disorders like celiac disease,26 with an associated poorer outcome.26 This effect has also been confirmed for T1D associated with zonulin expression, with those carrying the Hp2 variant at greater risk and suffering poorer outcomes (R).
However, there is currently no way to determine your HP status using commercial kits such as 23andMe as there are no polymorphisms associated with either type. As such the only way to confirm status would be to undergo sequencing for this gene, which is unfortunately a much more expensive proposition.
How to check your zonulin levels
There are a couple of ways to check your zonulin levels. The first is the most obvious and easy to perform, a simple blood test to check for levels of protein expression in the blood, as was used in the study investigating zonulin and celiac disease.
A second more complex option is the lactulose or mannitol test which assess the permeability of the gut by measuring the levels of both compounds in urine after they’re ingested. The higher the detected levels the leakier the gut.
There are a couple of issues with these tests however, the main one being that there are no accepted levels that can be considered good or bad, and as we know from this blog there can be a great degree of variability in peoples levels of compounds and their responses as well. So proceed with caution in relation to testing, it may be of use to track your gut function over time but don’t rely on the tests for diagnoses.
So to summarize, zonulin is an important modulator of gut permeability which is strongly associated with several disorders of the gut, with other potential associations as well. As a relatively new protein (it was only described in 2000) the science is still developing and genetic markers, diagnostic tests and therapeutic targets are still lacking. However, with improvements in testing this is clearly going to become an important biomarker for various disorders, we’re just not there yet.