8/25/2020 The Clinical Utility of Zonulin Testing | Kresser Institute

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.

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 istockphoto.com/g-stockstudio
permeability, and alternatives to zonulin testing.

Conte nts [hide]

What Is Zonulin?
Conditions Associate d with Ele v ate d Zonulin
Common Issue s with Zonulin Studie s
Alte rnativ e s to Zonulin Te sting
Is It Ev e n Worth Te sting for Le aky Gut?

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).

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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 hereto 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

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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!

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