Tight junctions (TJs) — impermeable (normally) connections between the cells lining our intestinal tracts — are critical for protecting us from environmental dangers such as toxins, bacteria and viruses. When TJs are disrupted, food particles and microorganisms can cross over directly into our circulation, causing widespread inflammation and contributing to a number of diseases. Zonulin disrupts specific proteins in our intestinal tracts that help maintain TJs and prevent a “leaky gut” (i.e., intestinal permeability), essentially poking microscopic holes in our intestinal lining. While its presence in the bloodstream has been used as an indicator of intestinal permeability and has been linked to several metabolic disorders, research is still controversial — and many don’t believe the alien-sounding protein is a marker of disease at all.

What is zonulin?

Zonulin was first described 12 years ago in a landmark paper that discovered a human protein almost identical to a toxin called “zonula occludens toxin (ZOT),” which is produced by the type of bacteria that cause cholera (acute, severe diarrhea). Researchers were interested in ZOT due to its ability to disrupt TJs and cause intestinal permeability, which has been linked to several chronic diseases.

Over 50 proteins help build and support TJs to ensure they work correctly (i.e., letting water, nutrients, and small particles important for training the immune system to pass through, but keeping everything else in the intestines). One of those proteins is called zonula occludens (there are actually several, and they are called ZO proteins), and it is a scaffold protein, meaning that it helps hold TJs together. When we accidentally ingest ZOT-producing bacteria like Vibrio cholerae, the toxin causes a structural change to the ZO scaffold proteins, which opens TJs. This, in turn, leads to the severe diarrhea associated with Vibrio cholerae infection. While not fun for us, it is critical for flushing out the pathogen. 

It was while studying the bacterial ZOT protein that researchers discovered a protein in human cells that is nearly identical, and they named it zonulin (importantly, it is also nearly identical to another human protein called prehaptoglobin-2, but we’ll get to that later). Zonulin, which is produced by intestinal and liver cells, is the only known modulator of TJs and is therefore a key player in the mucosal immune response. And while it can play a beneficial role — helping flush out pathogenic bacteria like Vibrio cholerae, if the normally tight control of zonulin levels is lost, our health can suffer severe consequences.

Can zonulin cause disease?

Researchers have been studying the connection between zonulin and disease for over a decade, and they consider increased zonulin levels in the bloodstream to be an indicator of an impaired intestinal barrier. Because an impaired intestinal barrier is also a characteristic of several metabolic and immune disorders, many studies have examined whether increased zonulin levels are characteristic of these diseases, too. Increased zonulin levels in the bloodstream have been linked to several chronic diseases:

• Celiac disease and gluten sensitivity: Several studies have connected increased zonulin and celiac disease. A recent study of kids with celiac disease suggested that changes in gut microbiome composition led to dysregulation of the normally tightly controlled zonulin pathway. The resulting “leaky gut” removed the body’s ability to recognize gluten as harmless, causing inappropriate immune responses to the food particle. Importantly, these children also had type 1 diabetes, which is tied to a higher genetic risk for celiac disease. In another study, individuals with celiac disease and gluten sensitivity had elevated zonlin in their blood, even more so than individuals with irritable bowel disease (IBD). A gluten-free diet relieved symptoms only in gluten sensitive individuals with a genetic predisposition for celiac.

• Type 1 and type 2 diabetes: Diving deeper into the connection between zonulin and diabetes, one study showed that increased zonulin in the bloodstream was associated with complications of diabetes, and they showed direct associations between zonulin and insulin resistance and lipid profile. Another study in children suggested that zonulin in the bloodstream is a potential biomarker of the risk for shared type 1 diabetes and celiac disease. Studies have also connected increased lipopolysaccharide (LPS) and zonulin with type 2 diabetes, demonstrating a positive correlation between these molecules, inflammatory markers and poor glycemic and lipid control. Similar results connecting zonulin with dyslipidemia, insulin resistance and inflammation were reported in a cohort of Chinese individuals with type 2 diabetes.

• Gestational diabetes: One study showed an association between increased zonulin in the bloodstream during early pregnancy and gestational diabetes, suggesting that zonulin could potentially be used as an early predictor for this disease.  

• Obesity: Cross-sectional studies have identified associations between zonulin and obesity and high energy intake, as well as with co-morbidities of obesity including fatty liver disease, insulin resistance and diabetes (see above). Interestingly, some of these studies have connected gut microbiome-related systemic inflammation in obese individuals with levels of zonulin in the blood. Additionally, in at least one study, weight loss interventions, including bariatric surgery, decreased zonulin in the blood and improved markers of glucose intolerance and liver disease.

• Inflammatory bowel disease (IBD): In a study published earlier this year, scientists for the first time identified an association between increased levels of zonulin in the blood with prevalent (i.e., existing disease) but not incident (i.e, new cases) IBD. Because zonulin was increased only in prevalent IBD, the study authors suggest that imparied intestinal permeability is a consequence, and not a cause, of the chronic inflammation that is characteristic of IBD. Nevertheless, the study was small and results are preliminary.  

Other studies have expressed a more conservative outlook on zonulin and disease. A 2017 study suggested that increased zonulin in the blood is a better marker of pre-disease and/or disease-associated factors related to metabolic syndrome and low-grade inflammation than it is a tool for predicting specific disease states:

• Higher zonulin levels in the blood were associated with higher waist circumference, higher blood pressure, higher blood glucose levels and increased risk of overweight, but not with gastrointestinal (GI) symptoms or incidence of GI disease. These results suggest zonulin may be a better marker of obesity, metabolic syndrome and low-grade inflammation than of impaired intestinal permeability. 

• These results are preliminary and studies haven’t determined the degree of intestinal permeability associated with obesity and metabolic syndrome or identified the reason behind elevated zonulin levels during these conditions. 

One common theme across all of these studies is that the relationship between zonulin and disease appears to be complex and also dictated by genetic risk, other environmental factors (such as the microbiome and diet) and even other metabolic or autoimmune diseases. Importantly, none of these studies demonstrate a cause-and-effect relationship between zonulin and disease. In fact, scientists don’t yet even know the degree to which zonulin and inflammation and zonulin and intestinal permeability are correlated. Some studies have even shown contradictory results to the studies discussed above. One reason may be due to the way scientists have been measuring zonulin in the blood.

Have we been measuring zonulin all wrong?

As we discussed earlier in this article, human zonulin is very similar to the bacterial toxin ZOT and also to another human protein called pre-haptoglobin 2 (preHP2). The way zonulin levels in the blood have historically been measured is through a molecular test called an ELISA. ELISA stands for enzyme-linked immunosorbent assay, and it works like this:

• The antigen, or the target (in this case zonulin, or more accurately, blood that might contain zonulin), is attached to a solid surface.
• An antibody to zonulin is linked to an enzyme (a protein that causes a reaction to happen) called a reporter enzyme. 
• The antibody-reporter enzyme link, and the substance the reporter enzyme works on, are both added to the solid surface.
• A color change occurs when the enzyme acts on its target substance. This reaction will only occur if the antibody-enzyme link binds to the antigen. So, if a color change is observed, zonulin is present. The degree of color change is directly related to the amount of zonulin present.

The ability of ELISA to work correctly depends on how well the antibody binds to the protein you want to measure. Last year, scientists revealed that commercial ELISA assays traditionally used to detect zonulin were using an antibody that mistakenly didn’t actually target the human zonulin protein. These scientists suggested that other ways to measure zonulin are desperately needed, and that in the meantime, researchers should measure intestinal permeability in other ways, such as by measuring ZO proteins (the proteins disrupted by zonulin).

Key takeaways

Zonulin is part of a tightly controlled system that can benefit us by removing disease-causing bacteria from our systems but can also harm us by increasing intestinal permeability. Several studies have linked increased zonulin to chronic diseases including diabetes, obesity and IBD — although inconsistencies across studies, confounding factors such as environment and genetics and errors in scientific assays make the connection between zonulin and these diseases cloudy at best. Further studies are needed to fully understand the relationship between zonulin and disease, and whether reducing zonulin through drugs or diet could help relieve disease symptoms.