The Clinical Utility of Zonulin Testing

In recent years, intestinal permeability, or "leaky gut," has been linked to an increasing number of chronic inflammatory diseases, and zonulin, a small protein produced in the gut, has emerged as a potential biomarker. But is there any use in diagnosing someone with leaky gut? Read on to learn about the utility of zonulin testing and other measures of intestinal permeability in clinical practice.

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Just a decade or two ago, anyone mentioning intestinal permeability was scoffed at and labeled a quack. Today, intestinal permeability is widely recognized in the scientific literature as a very real biological phenomenon and has been associated with a long list of chronic inflammatory conditions, including IBD, IBS, liver disease, autism, and eczema, to name a few (1, 2, 3, 4, 5). As a result, interest in testing for intestinal permeability in a clinical setting has risen dramatically, and several laboratories now offer tests.

One test that has grown rapidly in popularity is the measurement of serum or plasma zonulin. In this article, I’ll discuss what zonulin is, which conditions are accompanied by elevated zonulin, why I don’t trust it as a clinical marker of intestinal permeability, and alternatives to zonulin testing.

What Is Zonulin?

In the intestine, the epithelial cells that form the gut wall are connected to adjacent epithelial cells by protein complexes called tight junctions. These tight junctions consist of proteins including claudins, occludins, and E-cadherin, among others.

Zonulin testing is not reliable for intestinal permeability and it may not even be worth testing for leaky gut

In 2000, researcher Alessio Fasano and his team discovered zonulin, a protein that modulates tight junction integrity (6). Upon insult to the intestine, zonulin is released from epithelial cells, where it can act locally on receptors on the apical (lumen-facing) membrane of nearby cells. This starts a signaling cascade that leads to tight junction disassembly. Gliadin, a protein found in wheat, and bacterial exposure are particularly potent stimulators of zonulin release (7, 8).

Why would our guts produce a molecule that increases intestinal permeability? While the physiological roles of the zonulin system remain to be uncovered, zonulin is thought to be potentially involved in intestinal innate immunity. In 2009, it was identified as the precursor to haptoglobin-2 (HP2) (9). The only known function of haptoglobins to date is the binding of hemoglobin (Hb) to form stable HP–Hb complexes, preventing Hb from inducing oxidative tissue damage (10).

Conditions Associated with Elevated Zonulin

Despite our lack of knowledge of its normal role in the gut, zonulin has been used as a biomarker of impaired gut barrier function in autoimmune, neurodegenerative, and metabolic diseases. Serum or plasma zonulin has been shown to be elevated in autism (11), depression (12), type 1 diabetes (13), type 2 diabetes (14), metabolic syndrome (15), and PCOS (16). It is also associated with systemic inflammation and indices of physical frailty in aging (17) and is particularly elevated in those with celiac disease (6).

The basic idea is that with zonulin, a fairly large (47 kDa) protein produced in the gut, should not be able to pass into the bloodstream under normal conditions. Thus, its recovery in plasma or serum likely reflects the degree of intestinal permeability. While the theory makes sense, any good biomarker worth its predictive value will also have the following characteristics:

• Sensitivity
• Specificity
• Robustness
• Accuracy
• Reproducibility

In the next several sections, I’ll discuss why zonulin doesn’t hold up to these standards.

Common Issues with Zonulin Studies

First, in many of these studies I listed in the previous section linking elevated zonulin with disease, only a fraction of the patients with the condition showed elevated levels of zonulin. For example, a close look at the data from one study finds that only 42 percent of the patients with type 1 diabetes had an elevated serum zonulin level (defined as 2 standard deviations above the control group mean) (13). Another study found that of 30 patients with celiac disease, only 33 percent (10/30) had elevated serum zonulin (18).

Additionally, a recently published paper suggests that many of the canonical studies used to support the clinical utilization of zonulin were not measuring zonulin at all! Enzyme-linked immunosorbent assay (ELISA) is a common, relatively inexpensive technique to quantify protein levels in a sample. Using the more lucrative mass spectrometry, Scheffler et al. discovered that a popular ELISA kit from Germany was mainly capturing the zonulin analog properdin (19). The authors conclude:

“The Immundiagnostik ELISA kit supposedly testing serum zonulin (pre-HP2) levels could identify a variety of proteins structurally and possibly functionally related to zonulin, suggesting the existence of a family of zonulin proteins …”

Whether other kits have the same issue is unknown, but these findings definitely call the accuracy of zonulin testing into question. (Click here to download a full list of studies that used this ELISA kit; open access provided by Scheffler et al.)

Weak Correlations with Other Intestinal Permeability Measures

The legitimacy of zonulin testing can also be assessed by comparing it to other, well-validated methods. The differential sugar absorption test is considered the “gold standard” method for functional small intestinal permeability testing. Under normal conditions, large oligosaccharides like lactulose should not be able to traverse the intestinal barrier, while small monosaccharides like mannitol or rhamnose should be able to pass across freely. The test therefore involves consuming a solution containing these two differentially absorbed carbohydrates. Several hours later, the sugars are measured in the blood or urine, and the ratio of lactulose to mannitol or lactulose to rhamnose recovered reflects the degree of intestinal permeability.

Sapone et al. performed both zonulin and lactulose–mannitol testing and found only a very weak correlation between the results of the two assessments (correlation coefficient = 0.36) (13). The authors write: “While these numbers are very useful in the setting of research analysis, they are insufficiently correlated to be applied to diagnostic medical use.”

Low Reproducibility of Plasma Zonulin

To measure reproducibility, one study published recently in the World Journal of Gastroenterology analyzed 70 blood samples from 18 healthy human volunteers at intervals of zero, six, 24, and 30 hours (18). In a third of the participants (6/18), serum zonulin levels were so low that they were close to the detection limit of the assay. In this group, serum zonulin was fairly stable over time. For the other two thirds of the participants (12/18), serum zonulin levels fluctuated significantly between blood samples.

Another study on ICU patients with sepsis found that plasma zonulin values varied greatly from one day to the next (20). Taken together, these studies suggest that a single measurement of serum zonulin, as is performed by most clinical laboratories, may not be a reliable indicator for assessment of intestinal permeability. This may be because zonulin has a short half-life in the blood and is rapidly cleared by the immune system. Fecal zonulin has also been explored as a marker, but the stability of this marker over time is unknown and it may very likely mirror the fluctuations seen with blood zonulin.

Zonulin Antibodies in Blood

Though zonulin is cleared rapidly from circulation, the presence of zonulin in the blood triggers an immune response and the production of zonulin-specific antibodies. When Vojdani et al. measured IgG and IgA antibody levels against zonulin at the same time points (zero, six, 24, and 30 hours), they found that serum zonulin was somewhat correlated with anti-zonulin IgG and highly correlated with anti-zonulin IgA. Moreover, these antibody levels were determined to be “highly stable with variations of less than 10 percent” (18).

In celiac disease patients, 67 percent (20/30) produced antibodies to zonulin, compared to just 10 who presented with elevated zonulin. The authors therefore recommended the use of anti-zonulin antibodies for assessment of intestinal permeability in future studies (18). This is not the first time that antibody levels against an antigen have been found to be more stable and diagnostically useful than levels of the antigen itself (21).

Alternatives to Zonulin Testing

While it’s clear that zonulin is not a reliable test for intestinal permeability, there are some alternatives to zonulin testing:

• Differential sugar tests: As discussed above, DST is the most widely accepted and clinically validated method for assessing small intestinal permeability. However, GI motility, gastric emptying, and other factors can affect uptake of these sugars, and it is much more time consuming for the patient than a simple blood test. Additionally, lactulose–mannitol tests are useful only for assessing SI permeability, since lactulose is degraded by bacteria in the large intestine. To measure colonic permeability, a third sugar, such as sucralose, which is unaffected by colonic bacteria, is added to the mixture. The lactulose excretion over 24 hours is subtracted from the sucralose excretion to calculate an isolated measure of colonic permeability.
• Antigenic permeability screen: This method looks at antibodies against several molecules involved in intestinal permeability, including zonulin, tight junction proteins, and LPS. Cyrex Laboratories provides this test as Array 2, which includes actomyosin IgA and LPS, occludin, and zonulin IgG, IgA, and IgM. This test allows a clearer look at intestinal immune dysregulation, and I’ve found it to be much more useful than other measures.

Is It Even Worth Testing for Leaky Gut?

It’s also worth asking whether we should even bother testing for intestinal permeability. While intestinal permeability certainly contributes to disease, intestinal permeability itself is almost always caused by something further upstream. This could be food intolerances, SIBO, nutrient deficiencies, gut infections, chronic stress, and/or immune dysregulation, depending on the patient.

We’ll want to address these factors regardless of whether a patient has a leaky gut or not, since these underlying causes can affect many aspects of their health. Therefore, in general, I believe it’s more useful to test for these underlying pathologies than to test for leaky gut itself. If the patient’s symptoms don’t resolve with treatment, then I might order an antigenic permeability screen or a lactulose–mannitol test to determine the extent of GI pathology.

There are also some patients who are extremely motivated by an abnormal test result. If a patient isn’t convinced that some of the foods they are eating are problematic, for instance, or that their stress levels are impacting their gut health, testing for leaky gut could be useful, if only to motivate behavior change. Given the issues discussed in this article, though, I think it’s clear that zonulin testing is not the way to do this!