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Longevity Library · Lab reference

Written by Daniel Tagge, MD
Updated October 23, 2026
Reviewed October 24, 2026
40 sources · Measured in ng/mL

System: Gut-Immune Health

ZonulinSerum zonulin · Stool zonulin · Fecal zonulin · Pre-haptoglobin 2 · Prehaptoglobin-2

Zonulin is a real protein that loosens the tight junctions between gut lining cells, identified in 2009 as pre-haptoglobin 2, but the commercial blood and stool tests sold under its name have been shown by two independent laboratories not to detect it, their values do not track the research test of gut permeability, and no study links a measured zonulin to any outcome, so a zonulin result cannot diagnose a leaky gut or guide treatment.

Zonulin was discovered as the human counterpart of a cholera toxin that opens the seals between intestinal cells, and gluten and bacteria trigger its release in laboratory studies. The test that reached the catalog is another matter: the most widely used kit does not recognize pre-haptoglobin 2, it reports values in people who cannot make zonulin, and its results correlate poorly with the lactulose-mannitol test researchers use. The permeability science is worth taking seriously; this number is not yet a way to measure it.

01 · The scale

Where the laboratory range and the evidence sit.

No study ties a measured zonulin to any outcome, and no trial shows that acting on one helps anyone. The commonest commercial assay has been shown by two independent laboratories not to detect zonulin (pre-haptoglobin 2), and in three studies its values did not correlate with the lactulose-mannitol test of permeability. The associations in the literature, with obesity, insulin resistance and type 1 diabetes, are cross-sectional, mostly on that assay, and serum zonulin did not track gastrointestinal symptoms in two populations. The drug designed to close the zonulin-opened junction did not change measured permeability in two trials. The permeability biology is real; the number is a research marker whose validity is disputed.

0255075100

  1. Reference intervals are set by each laboratory on its own kit, serum and stool separately; none is outcome-derived, and no cited source supports any boundary on this scale, so the page draws none

    0 and above ng/mL · This entry, 2026; Massier 2021; Ajamian 2019

  • Laboratory range
Scale in ng/mL. Serum and stool zonulin are measured on different kits with their own ranges, and the two values are not interchangeable. Assays from different makers that claim to detect the same protein agreed poorly when run on the same blood, and two independent laboratories found that the most widely used kit does not detect zonulin (pre-haptoglobin 2) at all, so what the number measures is disputed before any range is applied. Bands show where studies found something, not a recommendation for any one person.

02 · What it is

What Zonulin measures.

Zonulin is a protein that opens the tight junctions, the seals between the cells lining the small intestine. It was found by looking for a human version of the zonula occludens toxin of Vibrio cholerae, which opens those seals: antibodies against the toxin picked out a protein from fetal and adult human intestine that took tight junctions apart in primate gut tissue mounted in a chamber, and the 2000 report in the Lancet found it raised in intestinal tissue during the acute phase of celiac disease, a condition in which the junctions are open and permeability is increased. Its discoverer describes it as the only physiological modulator of intercellular tight junctions described so far.1,2,3Grade C

In 2009 a proteomic analysis of human serum identified zonulin as the precursor of haptoglobin 2, the uncut single-chain form of a protein whose mature, cut form scavenges free hemoglobin. The uncut form carries a growth-factor-like segment that activates the epidermal growth factor receptor through a second receptor, PAR2, and that signal increased intestinal permeability; cutting the precursor into its two subunits abolished the effect, and mice lacking PAR2 kept their barrier. The authors called it the first example of a molecule whose precursor has a biological activity distinct from its mature form. Because only the haptoglobin 2 gene variant makes it, people who carry two copies of haptoglobin 1 make no zonulin at all.4,5Grade C

What a clinician means by gut permeability is measured in research by a sugar test, not a blood protein. In the dual-sugar test a person drinks two sugars of different sizes, typically lactulose and mannitol, and the ratio excreted in the urine over the following hours reports how much leaked across the small intestine; the method already had a 20-year history when its strengths and shortcomings were reviewed in 1995, and critics of the zonulin assay recommend it as the rigorous test to use instead. Even that test is noisy: in a 2012 trial its readings were so variable in outpatients that the investigators could not assess the drug's effect on permeability. The barrier is more than the tight junction, with surface mucus, the epithelium and immune defenses all contributing, and the 2019 review in Gut concludes that assessing it requires measurements beyond the epithelial layer.6,7,8,9Grade B

03 · Origins

Where it came from, and where it sits in the network.

The intestine has a problem no other surface in the body has on the same scale. A 2014 review describes it as a huge mucosal surface where billions of bacteria face the body's largest immune system: the barrier must keep microbes and toxins out and must stay open to absorb fluids and nutrients. The answer is a lining of epithelial cells whose seals, the tight junctions, are selectively permeable, opened physiologically by nutrients in the gut and pathologically by immune cells, cytokines, the enteric nervous system and pathogens. Together with the gut's lymphoid tissue and its nerve and hormone network, that paracellular gate sets the balance between tolerance of and immunity to what is eaten.10,11,12,13Grade C

The discovery began with a pathogen. Vibrio cholerae makes a toxin that binds a receptor on gut cells and sets off a cascade that loosens the tight junctions, and the Maryland group reasoned that a toxin this specific was probably imitating a molecule the host already made; that is how zonulin was found. In laboratory studies the host's own trigger looks defensive: small intestines from several species released zonulin from their luminal side when exposed to enteric bacteria, pathogenic or not, and the junctions opened, which the authors proposed may flush microorganisms out and help the host resist bacterial colonization of the small intestine.1,14Grade C

Gluten is the other trigger studied. Gliadin, a component of gluten, binds the chemokine receptor CXCR3 on intestinal cells and releases zonulin through the MyD88 pathway, increasing permeability in normal mouse intestine but not in intestine lacking the receptor. Biopsies from people with celiac disease in remission showed a sustained zonulin release and rise in permeability when exposed to gliadin, while biopsies from people without celiac disease showed a limited, transient release and a rise that never reached the celiac level; the authors concluded that gliadin activates zonulin signaling whatever a person's genetic risk for autoimmunity.15,16Grade C

The molecule is a recent and human-specific one. Haptoglobin is conserved in most mammals, but the haptoglobin 2 genotype is unique to humans, and zonulin as pre-haptoglobin 2 is not naturally expressed in mice; a letter in Gut points out that this makes zonulin measurements in ordinary mouse models highly questionable, since a mouse has no zonulin to measure. The animal work that built the permeability field therefore cannot be read as zonulin work unless the mice were engineered to carry human haptoglobin 2.9,4Grade C

The discoverer built a large hypothesis on the pathway: that when zonulin is deregulated in genetically susceptible people, the excess flow of foreign antigens through the barrier can cause intestinal and extraintestinal autoimmune, inflammatory and malignant disease, and that these processes might be arrested by restoring the barrier. A 2016 review calls zonulin a master regulator of intestinal permeability, and a 2020 review, titled All disease begins in the (leaky) gut, links the zonulin family to autoimmune, infectious, metabolic and tumoral disease within the hygiene hypothesis. These are reviews by the group that found the protein, and they state a model, not a demonstration.17,13,18,19Grade C

Where permeability sits in the network is downstream of diet and microbes and upstream of inflammation, as a hypothesis with some evidence. In mice, four weeks of a high-fat diet raised plasma bacterial lipopolysaccharide (endotoxin) two- to threefold, a state the authors named metabolic endotoxemia; infusing that much endotoxin reproduced the weight gain and raised fasting glucose and insulin, and mice lacking the endotoxin receptor CD14 resisted most of it. In 8 healthy people, a month on a Western-style diet raised endotoxin activity in the blood by 71 percent and a prudent diet lowered it by 31 percent. A 2016 review across many diseases concludes that no direct cause-and-effect relationship between permeability and inflammation has been confirmed. The neighbors on this shelf measure different things: fecal calprotectin reports inflammatory cells in the bowel wall, and hs-CRP reports inflammation anywhere in the body.20,21,22Grade C

04 · The laboratory range

What a laboratory calls normal.

Laboratory-specific: each kit maker and laboratory sets its own serum or stool interval, none is derived from outcomes, and because the most used assay's target is disputed, the question of what the kit detects comes before any range.

No published reference interval for zonulin is tied to health or disease. The printed range on a report is the distribution the kit maker or laboratory found in its own reference samples. Studies that divide people into high and low groups use their own medians: one cross-sectional study of 100 overweight women in early pregnancy split them at its median of 46.4 ng/mL, a cut chosen for the analysis, not a clinical threshold.23,9Grade C

The assay question comes first. Two commercial serum assays sold to detect the same protein, run on the sera of 142 people, correlated weakly and not significantly (Spearman's r 0.29, p 0.14); neither reliably detected recombinant pre-haptoglobin 2 added as a positive control; and the assays returned values in people whose haptoglobin type means they make no zonulin. Mass spectrometry of what the assays captured matched haptoglobin and complement C3 for one kit and complement C3 for the other, and the authors advised caution in using serum zonulin as a marker of barrier integrity until assay methods improve.5Grade C

Serum and stool are separate measurements. Studies that measure both report them separately, and in a 12-week diet study in 30 adults with type 2 diabetes, serum and fecal zonulin were measured on separate assays and reported separately: both rose by week 12, and the serum value was still raised at week 28. Nothing in the published literature establishes a conversion between the two, and a stool value cannot be read against a serum range or the reverse.24Grade C

05 · The evidence

What the studies support.

The decisive finding is about the test, not the protein. In 376 people from a well-characterized German cohort, zonulin measured on a widely used commercial ELISA did not correlate with haptoglobin genotype, which it should have if it detected pre-haptoglobin 2. Antibody capture, mass spectrometry and Western blotting found that none of the proteins the kit captured corresponded to pre-haptoglobin 2; the kit did not detect added recombinant pre-haptoglobin 2; and the most likely target was properdin, a complement protein the authors proposed as a possible second member of the zonulin family. Whatever the kit measured was higher in diabetes and obesity and tracked lipid and glucose markers. A second group, working independently, reached the same conclusion about two commercial assays the following year.25,5Grade B

A 2021 letter in Gut drew the consequence: measurements on the commercial ELISA do not reflect zonulin but concentrations of unknown proteins, which precludes conclusions about zonulin and permeability from those studies, positive or negative, and applies retrospectively to the many studies that used the kits. The letter tabulated three studies comparing the kit with the lactulose-mannitol test, in 38, 71 and 24 people, with correlations of 0.11, 0.17 and 0.033, none significant. Its authors stress that this does not take away from zonulin as a regulator of permeability, and they recommend dual-sugar tests and tissue studies until a specific assay exists.9Grade B

Where zonulin has been compared with symptoms, it did not sort them. In 363 Swedish adults, offspring of a cardiovascular cohort, neither gastrointestinal symptoms nor gastrointestinal diseases had any influence on serum zonulin, while higher values went with a larger waist, higher diastolic blood pressure and glucose, and more overweight, obesity and hyperlipidemia. An Australian study reported in Gut under the title that serum zonulin fails to identify irritable bowel syndrome, functional dyspepsia or non-coeliac wheat sensitivity found the same for those three conditions.26,27Grade C

The metabolic associations are cross-sectional and small. In 123 Caucasian men, circulating zonulin rose with body mass index, waist-to-hip ratio, fasting insulin, triglycerides, uric acid and interleukin-6 and fell with HDL cholesterol and insulin sensitivity; once interleukin-6 entered the model, only body mass index contributed independently. In 50 obese and 30 normal-weight adults, plasma zonulin was higher in the obese and correlated with age (r 0.43), body mass index (r 0.33) and fat mass (r 0.31). In 100 overweight pregnant women, those below the study's median had richer gut microbiota and ate more fiber and omega-3 fats. All three used commercial ELISAs; whether they measured zonulin is the open question the assay studies raised.28,29,23,25Grade C

The type 1 diabetes finding is the most cited and comes from the discoverer's group. Among 339 patients with type 1 diabetes, 141 (42 percent) had serum zonulin above that of age-matched controls; higher zonulin went with higher permeability measured in the body and with changes in tight junction gene expression; and in stored serum from the years before diagnosis, raised zonulin was found in 70 percent and preceded onset by about 3.5 years in those who went on to develop the disease. It has not been reproduced on an assay shown to detect pre-haptoglobin 2, and no study has acted on it.30Grade C

The drug trials show the pathway is a plausible target and that closing it did not do what the model predicted. Larazotide acetate is an oral peptide designed to prevent tight junctions from opening. In 86 people with diet-controlled celiac disease given gluten for 14 days, the lactulose-mannitol ratio was too variable to show any effect of the drug, though some lower doses limited symptoms. In 184 people given gluten for 6 weeks, the ratio did not differ between drug and placebo, while the lowest dose limited symptoms and all doses blunted the rise in tissue transglutaminase antibodies. In 342 people with symptoms despite a gluten-free diet, the lowest dose met the symptom end point and the two higher doses were no different from placebo. Symptom signals at the lowest dose only, with no measured change in permeability, are not the result a barrier drug was expected to give.8,31,32Grade B

The independent reviews agree on the limit. A 2013 review found most data on permeability and disease correlative, noted that barrier loss alone was insufficient to start disease in human and mouse studies, that it is uncertain whether increased permeability is cause or effect, and that no barrier-restoring treatment had been proven, so that treatment should target the underlying disease. The 2019 review in Gut adds that no inflammatory or ulcerating intestinal disease can be cured by normalizing barrier function, that it is still unproven that restoring the barrier improves gastrointestinal or systemic disease, and that claims about a leaky gut in the public domain need confirmation before anyone endorses dietary exclusions or supplements to repair it.11,7,10Grade B

Celiac disease is the one condition on this page with a guideline, and the guideline does not use zonulin. The 2023 American College of Gastroenterology update screens with celiac-specific antibodies such as tissue transglutaminase in the serum and confirms with intestinal biopsy in most patients, and treats with a strict, lifelong gluten-free diet. A raised zonulin is not a test for celiac disease, and a normal one does not exclude it.33Grade A

06 · What moves it

The levers with evidence behind them.

What is listed here has been tested. The size of each effect, and the size of the study, are in the sentence, so a small effect reads as a small effect.

  • Nonsteroidal anti-inflammatory drugs

    raise small intestinal permeability, measurably, within days

    NSAIDs frequently cause inflammation of the small intestine, with blood and protein loss, and occasionally ulcers, strictures and perforation, a range of damage distal to the duodenum that may be silent. In 23 healthy volunteers, indomethacin used as a positive control more than doubled the two-hour lactulose-mannitol ratio (0.071 against 0.030). The 2019 Gut review lists NSAIDs among the stresses that reliably increase permeability. This is the lever with the clearest evidence, and it acts on the barrier, not on the zonulin number.34,35,7Grade B

  • Alcohol

    raises permeability in heavy drinkers, recovering over days to weeks

    In 36 alcoholic patients without cirrhosis, those who had stopped drinking less than 4 days earlier almost always had higher small intestinal permeability than controls, and in many it stayed abnormal for up to 2 weeks after they stopped. In a later study, alcoholics with chronic liver disease had a markedly raised lactulose-mannitol ratio (0.703 against 0.019 in controls), while alcoholics without liver disease and people with liver disease who did not drink had normal ratios; the authors proposed that a leaky gut may be a necessary cofactor for liver injury in heavy drinkers. These are studies of heavy drinking, not of moderate intake.36,37Grade C

  • Prolonged or intense exercise

    raises permeability transiently, more with heat and running

    A systematic review found considerable evidence that intestinal injury, permeability and endotoxin in the blood rise with exercise intensity and duration, worse with heat stress and running, with about 2 hours at 60 percent of maximal oxygen uptake as the threshold at which significant disturbance appears, regardless of fitness, and called the impact major and reversible. In six trained volunteers, an hour at 80 percent of peak oxygen uptake raised small intestinal permeability compared with rest or easier running, without more gastrointestinal symptoms. Moderate exercise is not the concern; the long, hot, hard session is, and the barrier recovers.38,39Grade B

  • Acute psychological stress

    raises permeability, through mast cells

    In 23 healthy volunteers, a public speech raised the lactulose-mannitol ratio, but only in those whose salivary cortisol rose; an infusion of corticotropin-releasing hormone reproduced the effect, and a mast cell stabilizer blocked both the hormone's effect and the speech's. The authors concluded that acute psychological stress increases small intestinal permeability in humans by a mast cell mechanism. The study is small and acute; what chronic stress does over months is not measured.35Grade C

  • Overall diet pattern

    a Western pattern raised endotoxin in the blood in one small study

    In 8 healthy people, a month on a Western-style diet raised plasma endotoxin activity by 71 percent and a prudent diet lowered it by 31 percent; the mouse work that named metabolic endotoxemia found the same direction with a high-fat diet. The 2019 Gut review notes that dietary factors can reverse leakiness and mucosal damage in the stress states and that surfactants such as bile acids and food emulsifiers increase permeability. Endotoxin is not zonulin, and none of these studies tested a diet against an outcome through the barrier.21,20,7Grade C

  • Fiber and the gut microbiota

    associated with lower values in one study and raised them in another

    In 100 overweight pregnant women, those with zonulin below the study median had richer gut microbiota, more Faecalibacterium prausnitzii, and higher intakes of fiber and omega-3 fats. Yet when 30 adults with type 2 diabetes were served a diet with less carbohydrate and more fiber, fat and protein for 12 weeks, serum and fecal zonulin both rose. Both studies used commercial ELISAs. A number that moves in opposite directions with similar dietary changes, on an assay whose target is disputed, cannot tell anyone whether their fiber intake is helping their barrier.23,24,25Grade C

  • Gluten, in celiac disease

    a gluten-free diet normalized permeability in about half of treated patients

    In 21 adults with celiac disease on a gluten-free diet for a mean of 9.7 years, 10 had normal biopsies, normal permeability and normal antibodies; 6 had persisting intestinal damage, and all of those had an abnormal permeability test or antibody, possibly from continued gluten exposure. In laboratory tissue, gliadin opened the barrier strongly and lastingly in celiac mucosa and only briefly in non-celiac mucosa. For someone with celiac disease, removing gluten is the treatment; for someone without it, the evidence does not show a lasting barrier effect of gluten.40,16Grade C

07 · Reading it well

Caveats, and what belongs with a physician.

The test may not measure the protein its name promises. Two independent laboratories found that commercial kits do not recognize pre-haptoglobin 2, one identified properdin and the other complement C3 and haptoglobin among the captured proteins, and the kits reported values in people who make no zonulin. Every study built on those kits, including most of the associations on this page, reports something real about an unidentified protein and nothing settled about zonulin.25,5,9Grade B

Leaky gut is not an established stand-alone diagnosis. Intestinal barrier and intestinal permeability are poorly defined terms, their assessment is debated, and their clinical significance is not clearly established; the methods vary enormously and probably measure different parts of the barrier. A 2013 review traced the popularity of the leaky gut syndrome diagnosis to the correlation between permeability and disease and noted that its proponents' claim that barrier restoration will cure disease has not been shown in trials.10,11Grade B

Permeability is a moving physiological variable, not a fixed trait. It rises with endurance exercise, NSAIDs, pregnancy, acute stress, bile acids and food emulsifiers, and tight junctions open in response to nutrients in the gut as part of normal absorption. A single value on any test, sugar or protein, is a moment, and the reason for a raised one is usually in the history.7,11,35Grade C

Removing gluten before testing can hide celiac disease. Celiac disease is diagnosed by antibodies and, in most adults, a biopsy; on a gluten-free diet, antibodies, biopsy and permeability all normalized in about half of treated patients in one series. A person who stops gluten because of a zonulin result and is tested for celiac disease later may test normal while having the disease.33,40Grade B

The pathway's discoverer has declared a commercial interest. The 2009 paper identifying zonulin as pre-haptoglobin 2 discloses that its senior author and a co-author had a financial interest in a company developing treatments for celiac disease alternative to the gluten-free diet, the 2008 gliadin paper discloses a financial relationship with the same company, and the 2020 review discloses that its author is a co-founder and stockholder of a company developing treatments that exploit gut permeability. Disclosure is a fact about the papers, not a finding against them; it is a reason to give weight to the independent work, much of which is critical of the assay.4,15,19Grade C

See a physician

  • A zonulin result being used to recommend removing gluten, dairy or other foods, or to sell a supplement protocol, which is a reason to stop and talk to a physician first: the test's target is disputed and no trial supports acting on it.
  • Anyone thinking of going gluten-free because of gut or other symptoms, who should be tested for celiac disease first, while still eating gluten, because the antibody test and biopsy can return to normal on a gluten-free diet.
  • Diarrhea that wakes you at night, blood in the stool, unintended weight loss, anemia, or a family history of inflammatory bowel disease or bowel cancer, where the questions are answered by blood tests, fecal calprotectin and often endoscopy, not by zonulin.
  • Regular use of NSAIDs such as ibuprofen, naproxen or high-dose aspirin with abdominal pain, dark stools or iron deficiency, because these drugs can injure the small intestine silently.
  • Heavy drinking with gut symptoms or abnormal liver tests, where permeability and liver injury are linked in the research and the conversation is about alcohol.
  • Recurrent gut symptoms during long or hot endurance training or racing, which have their own management and do not need a zonulin test to explain them.

This page is education, not individual medical advice, and reading it creates no physician-patient relationship.

08 · Open questions

What the literature does not settle.

  • No assay shown to detect pre-haptoglobin 2 specifically is in routine use, and until one is, almost none of the clinical zonulin literature can be read as being about zonulin; whether a specific assay would tie serum zonulin to permeability or to disease is unknown.
  • Whether the properdin and complement proteins the commercial kits capture carry meaning of their own is open: what the kits measured did track obesity, diabetes and lipid markers, and no one knows why.
  • The type 1 diabetes finding, raised zonulin preceding onset by years, has not been reproduced on a validated assay or tested by intervention.
  • Whether increased permeability causes the conditions it travels with, or follows from them, is unresolved in every disease studied, and barrier loss alone did not start disease in the human and animal work reviewed.
  • The tight junction drug trials found symptom signals at the lowest dose and no measured change in permeability; whether a barrier drug can help anyone, and through what mechanism, is not established.
  • Stool zonulin has even less validation than serum zonulin, and no study has defined what a stool value means or how it relates to a serum value.
  • The dual-sugar test is the research standard but is variable in outpatients and not standardized across centers, so even the reference method for permeability is not ready for routine clinical use.

09 · In practice

How Dr. Tagge reads it in his own practice.

I take the gut barrier seriously as biology and I take the zonulin number lightly, and I want to be plain about why. The protein is real and the permeability research is good science, but the tests most laboratories sell under its name have been shown not to detect it, so I do not know what a zonulin result is telling me, and neither does anyone else yet. This is a research marker that reached the catalog before it reached the evidence. When someone asks me whether their gut is leaky, I answer the question through things I can interpret: calprotectin for inflammation in the bowel wall, the symptoms and their pattern, celiac antibodies done properly while gluten is still in the diet, and the history that moves the barrier, which is NSAIDs, alcohol, training load, stress and sleep. A single marker is a clue; several read against each other and against the person are a story, and zonulin is not yet a reliable clue. I do not diagnose leaky gut from a lab line and I do not sell a protocol against one. I will say where I am ahead of the evidence: I act on the levers that raise permeability in the studies, because they are worth changing for other reasons too, without proof that fixing the barrier changes any outcome.

A practice judgment, labeled as one (Grade D): it is how one physician reads the number for the people he cares for, not a recommendation for you.

10

Questions

11 · Sources

40 sources, read in full.

Numbered in the order they appear. Each line says what this page relies on from the paper; the link opens the record at PubMed or the publisher.

  1. 1.

    Wang W, Uzzau S, Goldblum SE, Fasano A. Human zonulin, a potential modulator of intestinal tight junctions. Journal of Cell Science. 2000. PMID 11082037. DOI 10.1242/jcs.113.24.4435.

    Laboratory method study · Antibodies against Vibrio cholerae zonula occludens toxin identified a novel human intestinal protein, zonulin, that induced tight junction disassembly in non-human primate intestinal epithelia in Ussing chambers; the toxin was postulated to mimic an endogenous modulator.

  2. 2.

    Fasano A, Not T, Wang W, Uzzau S, Berti I, Tommasini A, Goldblum SE. Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease. Lancet. 2000. PMID 10801176. DOI 10.1016/S0140-6736(00)02169-3.

    Laboratory method study · Zonulin, a human analogue of the Vibrio cholerae zonula occludens toxin, induces tight junction disassembly and increased permeability; its expression was raised in intestinal tissue during the acute phase of coeliac disease.

  3. 3.

    Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Annals of the New York Academy of Sciences. 2012. PMID 22731712. DOI 10.1111/j.1749-6632.2012.06538.x.

    Review · The discoverer describes zonulin as the only known physiologic modulator of intercellular tight junctions described so far, whose upregulation in genetically susceptible individuals leads to autoimmune disease.

  4. 4.

    Tripathi A, Lammers KM, Goldblum S, Shea-Donohue T, Netzel-Arnett S, Buzza MS, et al.. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proceedings of the National Academy of Sciences of the United States of America. 2009. PMID 19805376. DOI 10.1073/pnas.0906773106.

    Laboratory method study · Proteomics of human sera identified zonulin as pre-haptoglobin 2; its EGF-like motif transactivates EGFR via PAR2 to increase permeability, PAR2 silencing or knockout prevented barrier loss, and cleavage into alpha2 and beta subunits abolished the effect; authors disclosed a financial interest in a company developing celiac treatments alternative to the gluten-free diet.

  5. 5.

    Ajamian M, Steer D, Rosella G, Gibson PR. Serum zonulin as a marker of intestinal mucosal barrier function: May not be what it seems. PLoS One. 2019. PMID 30640940. DOI 10.1371/journal.pone.0210728.

    Laboratory method study · In 142 participants, two commercial zonulin assays correlated weakly (Spearman r 0.29, p 0.14), neither reliably detected recombinant pre-haptoglobin 2, values were reported in Hp1-1 zonulin non-producers, and mass spectrometry matched the captured antigen to haptoglobin and complement C3 or to complement C3; commercial assays are not detecting zonulin.

  6. 6.

    Bjarnason I, MacPherson A, Hollander D. Intestinal permeability: an overview. Gastroenterology. 1995. PMID 7729650. DOI 10.1016/0016-5085(95)90708-4.

    Review · Noninvasive permeability testing by differential urinary excretion of orally administered markers had a 20-year history by 1995; the review sets out its principles, uses, controversies over the permeation pathways, and the need for research into barrier regulation.

  7. 7.

    Camilleri M. Leaky gut: mechanisms, measurement and clinical implications in humans. Gut. 2019. PMID 31076401. DOI 10.1136/gutjnl-2019-318427.

    Review · The barrier includes mucus, epithelium and immune defenses and its assessment requires measurements beyond the epithelium; endurance exercise, NSAIDs, pregnancy and surfactants increase permeability; no ulcerating or inflammatory disease is cured by normalizing barrier function, benefit of barrier restoration is unproven, and public claims need confirmation before endorsing dietary exclusions or supplements.

  8. 8.

    Leffler DA, Kelly CP, Abdallah HZ, Colatrella AM, Harris LA, Leon F, et al.. A randomized, double-blind study of larazotide acetate to prevent the activation of celiac disease during gluten challenge. American Journal of Gastroenterology. 2012. PMID 22825365. DOI 10.1038/ajg.2012.211.

    Randomized trial · In 86 diet-controlled celiac patients randomized to larazotide acetate or placebo with or without 2.4 g/day gluten for 14 days, lactulose-mannitol variability in outpatients precluded assessing the drug's effect on permeability; some lower doses limited gluten-induced symptom worsening.

  9. 9.

    Massier L, Chakaroun R, Kovacs P, Heiker JT. Blurring the picture in leaky gut research: how shortcomings of zonulin as a biomarker mislead the field of intestinal permeability. Gut. 2021. PMID 33037053. DOI 10.1136/gutjnl-2020-323026.

    Review · Commercial zonulin ELISAs measure unknown proteins, not zonulin, precluding conclusions from studies that used them; in three studies (38, 71 and 24 people) the kit correlated with lactulose-mannitol at r 0.11, 0.17 and 0.033, none significant; the HP2 genotype is unique to humans and pre-haptoglobin 2 is not expressed in mice; dual-sugar tests are recommended instead.

  10. 10.

    Bischoff SC, Barbara G, Buurman W, Ockhuizen T, Schulzke JD, Serino M, et al.. Intestinal permeability--a new target for disease prevention and therapy. BMC Gastroenterology. 2014. PMID 25407511. DOI 10.1186/s12876-014-0189-7.

    Review · Intestinal barrier and permeability are poorly defined, their assessment is debated and their clinical significance not established; the barrier is a huge surface where billions of bacteria face the largest immune system and must both exclude microbes and absorb nutrients; assessment methods vary enormously and probably measure different components.

  11. 11.

    Odenwald MA, Turner JR. Intestinal permeability defects: is it time to treat?. Clinical Gastroenterology and Hepatology. 2013. PMID 23851019. DOI 10.1016/j.cgh.2013.07.001.

    Review · Tight junctions are selectively permeable and open physiologically to luminal nutrients and pathologically to immune cells, cytokines, nerves and pathogens; most data are correlative, barrier loss alone is insufficient to initiate disease, cause versus effect is uncertain, barrier restoration as cure has not been shown in trials, and treatment should target the underlying disease.

  12. 12.

    Turner JR. Intestinal mucosal barrier function in health and disease. Nature Reviews Immunology. 2009. PMID 19855405. DOI 10.1038/nri2653.

    Review · Mucosal epithelium must be a barrier against a hostile external environment and also absorb nutrients and secrete waste, which requires a selectively permeable barrier at the center of interactions between mucosal immunity and luminal contents.

  13. 13.

    Fasano A. Zonulin and its regulation of intestinal barrier function: the biological door to inflammation, autoimmunity, and cancer. Physiological Reviews. 2011. PMID 21248165. DOI 10.1152/physrev.00003.2008.

    Review · With gut lymphoid tissue and the neuroendocrine network, the epithelial barrier and its tight junctions control the balance between tolerance and immunity; deregulated zonulin in susceptible people is proposed to cause autoimmune, inflammatory and neoplastic disorders that might be arrested by restoring the barrier.

  14. 14.

    El Asmar R, Panigrahi P, Bamford P, Berti I, Not T, Coppa GV, et al.. Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure. Gastroenterology. 2002. PMID 12404235. DOI 10.1053/gast.2002.36578.

    Laboratory method study · Small intestines exposed to enteric bacteria secreted zonulin luminally regardless of species or bacterial virulence, followed by tight junction disassembly; the authors propose a defensive mechanism that flushes out microorganisms.

  15. 15.

    Lammers KM, Lu R, Brownley J, Lu B, Gerard C, Thomas K, et al.. Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3. Gastroenterology. 2008. PMID 18485912. DOI 10.1053/j.gastro.2008.03.023.

    Laboratory method study · Gliadin binds CXCR3 and causes MyD88-dependent zonulin release and increased permeability in wild-type but not CXCR3-knockout mouse intestine; mucosal CXCR3 was raised in active celiac disease; two authors disclosed a financial relationship with a company.

  16. 16.

    Drago S, El Asmar R, Di Pierro M, Grazia Clemente M, Tripathi A, Sapone A, et al.. Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines. Scandinavian Journal of Gastroenterology. 2006. PMID 16635908. DOI 10.1080/00365520500235334.

    Laboratory method study · Gliadin caused sustained zonulin release and permeability increase in biopsies from celiac patients in remission, and a limited, transient release with a smaller permeability rise in non-celiac biopsies; gliadin activates zonulin signaling irrespective of genetic risk.

  17. 17.

    Fasano A. Physiological, pathological, and therapeutic implications of zonulin-mediated intestinal barrier modulation: living life on the edge of the wall. American Journal of Pathology. 2008. PMID 18832585. DOI 10.2353/ajpath.2008.080192.

    Review · Proposes that prolonged zonulin upregulation lets excessive non-self antigens into the submucosa and causes intestinal and extraintestinal autoimmunity in susceptible people, and that the autoimmune process can be arrested by re-establishing barrier competency.

  18. 18.

    Sturgeon C, Fasano A. Zonulin, a regulator of epithelial and endothelial barrier functions, and its involvement in chronic inflammatory diseases. Tissue Barriers. 2016. PMID 28123927. DOI 10.1080/21688370.2016.1251384.

    Review · Review by the discoverer's group presenting zonulin as a master regulator of intestinal permeability linked to the development of several chronic inflammatory disorders.

  19. 19.

    Fasano A. All disease begins in the (leaky) gut: role of zonulin-mediated gut permeability in the pathogenesis of some chronic inflammatory diseases. F1000Research. 2020. PMID 32051759. DOI 10.12688/f1000research.20510.1.

    Review · Places increased permeability, an over-reactive immune system and the microbiome within the hygiene hypothesis for chronic inflammatory diseases and links the zonulin family to autoimmune, infective, metabolic and tumoral disease; the author discloses being co-founder and stockholder of a company developing treatments that exploit gut permeability.

  20. 20.

    Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, et al.. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes. 2007. PMID 17456850. DOI 10.2337/db06-1491.

    Laboratory method study · In mice, 4 weeks of high-fat diet raised plasma lipopolysaccharide two- to threefold (metabolic endotoxemia); infusing LPS reproduced weight gain and raised fasting glucose and insulin; CD14 mutant mice resisted most LPS- and diet-induced features.

  21. 21.

    Pendyala S, Walker JM, Holt PR. A high-fat diet is associated with endotoxemia that originates from the gut. Gastroenterology. 2012. PMID 22326433. DOI 10.1053/j.gastro.2012.01.034.

    Laboratory method study · In 8 healthy subjects, one month on a Western-style diet raised plasma endotoxin activity by 71 percent, while a prudent-style diet lowered it by 31 percent.

  22. 22.

    Fukui H. Increased Intestinal Permeability and Decreased Barrier Function: Does It Really Influence the Risk of Inflammation?. Inflammatory Intestinal Diseases. 2016. PMID 29922669. DOI 10.1159/000447252.

    Review · Increased permeability with endotoxemia and cytokinemia is described across many intestinal and systemic diseases, mostly assessed by sugar challenge tests or circulating bacterial markers, but no direct cause-and-effect relationship has been confirmed.

  23. 23.

    Mokkala K, Röytiö H, Munukka E, Pietilä S, Ekblad U, Rönnemaa T, et al.. Gut Microbiota Richness and Composition and Dietary Intake of Overweight Pregnant Women Are Related to Serum Zonulin Concentration, a Marker for Intestinal Permeability. Journal of Nutrition. 2016. PMID 27466607. DOI 10.3945/jn.116.235358.

    Cross-sectional study · In 100 overweight women in early pregnancy split at the median serum zonulin of 46.4 ng/mL (ELISA), the low group had richer microbiota, more Faecalibacterium prausnitzii, and higher intakes of n-3 fatty acids, fiber and several vitamins and minerals.

  24. 24.

    Ohlsson B, Roth B, Larsson E, Höglund P. Calprotectin in serum and zonulin in serum and feces are elevated after introduction of a diet with lower carbohydrate content and higher fiber, fat and protein contents. Biomedical Reports. 2017. PMID 28413639. DOI 10.3892/br.2017.865.

    Prospective cohort · In 30 adults with type 2 diabetes served an Okinawan-based Nordic diet (lower carbohydrate, higher fiber, fat and protein) for 12 weeks, serum and fecal zonulin rose at 12 weeks and serum zonulin remained raised at 28 weeks; uncontrolled before-and-after design.

  25. 25.

    Scheffler L, Crane A, Heyne H, Tönjes A, Schleinitz D, Ihling CH, et al.. Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family. Frontiers in Endocrinology. 2018. PMID 29459849. DOI 10.3389/fendo.2018.00022.

    Laboratory method study · In 376 Sorbs, ELISA zonulin did not correlate with haptoglobin genotype; none of the captured proteins was pre-haptoglobin 2, the kit did not detect recombinant pre-haptoglobin 2, and properdin was the most likely target; the measured protein was higher in diabetes and obesity and tracked lipid and glucose markers.

  26. 26.

    Ohlsson B, Orho-Melander M, Nilsson PM. Higher Levels of Serum Zonulin May Rather Be Associated with Increased Risk of Obesity and Hyperlipidemia, Than with Gastrointestinal Symptoms or Disease Manifestations. International Journal of Molecular Sciences. 2017. PMID 28282855. DOI 10.3390/ijms18030582.

    Cross-sectional study · In 363 adult offspring of a Swedish cardiovascular cohort, gastrointestinal symptoms and diseases had no influence on serum zonulin, while higher zonulin went with waist circumference, diastolic pressure, glucose, overweight, obesity and hyperlipidemia.

  27. 27.

    Talley NJ, Holtmann GJ, Jones M, Koloski NA, Walker MM, Burns G, et al.. Zonulin in serum as a biomarker fails to identify the IBS, functional dyspepsia and non-coeliac wheat sensitivity. Gut. 2020. PMID 31563879. DOI 10.1136/gutjnl-2019-318664.

    Cross-sectional study · Research letter reporting, as its title states, that serum zonulin failed to identify irritable bowel syndrome, functional dyspepsia or non-coeliac wheat sensitivity (no abstract; used for its title only).

  28. 28.

    Moreno-Navarrete JM, Sabater M, Ortega F, Ricart W, Fernández-Real JM. Circulating zonulin, a marker of intestinal permeability, is increased in association with obesity-associated insulin resistance. PLoS One. 2012. PMID 22629362. DOI 10.1371/journal.pone.0037160.

    Cross-sectional study · In 123 Caucasian men, ELISA zonulin rose with BMI, waist-to-hip ratio, fasting insulin, triglycerides, uric acid and IL-6 and fell with HDL and insulin sensitivity; with IL-6 in the model only BMI contributed independently.

  29. 29.

    Zak-Gołąb A, Kocełak P, Aptekorz M, Zientara M, Juszczyk L, Martirosian G, et al.. Gut microbiota, microinflammation, metabolic profile, and zonulin concentration in obese and normal weight subjects. International Journal of Endocrinology. 2013. PMID 23970898. DOI 10.1155/2013/674106.

    Cross-sectional study · In 50 obese and 30 normal-weight adults, plasma zonulin was higher in the obese and correlated with age (r 0.43), BMI (r 0.33), fat mass (r 0.31) and bacterial colony count (r 0.26).

  30. 30.

    Sapone A, de Magistris L, Pietzak M, Clemente MG, Tripathi A, Cucca F, et al.. Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes. 2006. PMID 16644703. DOI 10.2337/db05-1593.

    Cross-sectional study · 141 of 339 (42 percent) patients with type 1 diabetes had abnormal serum zonulin, which correlated with in vivo permeability; raised zonulin was present in 70 percent of pre-diabetes samples and preceded onset by 3.5 years in those who developed the disease.

  31. 31.

    Kelly CP, Green PH, Murray JA, Dimarino A, Colatrella A, Leffler DA, et al.. Larazotide acetate in patients with coeliac disease undergoing a gluten challenge: a randomised placebo-controlled study. Alimentary Pharmacology and Therapeutics. 2013. PMID 23163616. DOI 10.1111/apt.12147.

    Randomized trial · In 184 celiac patients given 2.7 g gluten daily for 6 weeks, lactulose-mannitol ratios did not differ between larazotide acetate and placebo; the 1-mg dose limited symptoms and all doses blunted the rise in anti-transglutaminase IgA (ratio 19.0 on placebo against 3.88 to 7.72).

  32. 32.

    Leffler DA, Kelly CP, Green PH, Fedorak RN, DiMarino A, Perrow W, et al.. Larazotide acetate for persistent symptoms of celiac disease despite a gluten-free diet: a randomized controlled trial. Gastroenterology. 2015. PMID 25683116. DOI 10.1053/j.gastro.2015.02.008.

    Randomized trial · In 342 adults with celiac symptoms despite a gluten-free diet, 0.5 mg larazotide acetate three times daily met the symptom end point; the 1- and 2-mg doses were no different from placebo on any end point.

  33. 33.

    Rubio-Tapia A, Hill ID, Semrad C, Kelly CP, Greer KB, Limketkai BN, Lebwohl B. American College of Gastroenterology Guidelines Update: Diagnosis and Management of Celiac Disease. American Journal of Gastroenterology. 2023. PMID 36602836. DOI 10.14309/ajg.0000000000002075.

    Guideline · Celiac-specific serum antibodies such as tissue transglutaminase are used for initial screening, intestinal biopsy confirms the diagnosis in most patients, and treatment is strict adherence to a gluten-free diet with lifelong follow-up.

  34. 34.

    Bjarnason I, Hayllar J, MacPherson AJ, Russell AS. Side effects of nonsteroidal anti-inflammatory drugs on the small and large intestine in humans. Gastroenterology. 1993. PMID 8500743. DOI 10.1016/0016-5085(93)90667-2.

    Review · NSAIDs frequently cause small intestinal inflammation with blood and protein loss and occasionally ulcers, strictures and perforation; damage distal to the duodenum may be asymptomatic or life threatening.

  35. 35.

    Vanuytsel T, van Wanrooy S, Vanheel H, Vanormelingen C, Verschueren S, Houben E, et al.. Psychological stress and corticotropin-releasing hormone increase intestinal permeability in humans by a mast cell-dependent mechanism. Gut. 2014. PMID 24153250. DOI 10.1136/gutjnl-2013-305690.

    Laboratory method study · In 23 healthy volunteers, indomethacin (lactulose-mannitol ratio 0.071 against 0.030) and public speech (0.059) raised permeability, the speech only in those whose cortisol rose; CRH raised it (0.042 against 0.028) and the mast cell stabilizer disodium cromoglycate blocked both CRH and speech effects.

  36. 36.

    Bjarnason I, Peters TJ, Wise RJ. The leaky gut of alcoholism: possible route of entry for toxic compounds. Lancet. 1984. PMID 6141332. DOI 10.1016/s0140-6736(84)92109-3.

    Cross-sectional study · In 36 non-intoxicated alcoholic patients without cirrhosis, those abstaining less than 4 days almost invariably had higher small intestinal permeability (chromium-51-EDTA) than controls, and in many it persisted up to 2 weeks after stopping.

  37. 37.

    Keshavarzian A, Holmes EW, Patel M, Iber F, Fields JZ, Pethkar S. Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage. American Journal of Gastroenterology. 1999. PMID 9934756. DOI 10.1111/j.1572-0241.1999.00797.x.

    Cross-sectional study · Alcoholics with chronic liver disease had a markedly raised urinary lactulose-mannitol ratio (0.703 against 0.019 in controls), while alcoholics without liver disease and non-drinkers with liver disease had normal ratios.

  38. 38.

    Costa RJS, Snipe RMJ, Kitic CM, Gibson PR. Systematic review: exercise-induced gastrointestinal syndrome-implications for health and intestinal disease. Alimentary Pharmacology and Therapeutics. 2017. PMID 28589631. DOI 10.1111/apt.14157.

    Review · Intestinal injury, permeability and endotoxemia rise with exercise intensity and duration, worse with heat and running; about 2 hours at 60 percent VO2max is the threshold for significant perturbation irrespective of fitness; the impact is major and reversible.

  39. 39.

    Pals KL, Chang RT, Ryan AJ, Gisolfi CV. Effect of running intensity on intestinal permeability. Journal of Applied Physiology. 1997. PMID 9049739. DOI 10.1152/jappl.1997.82.2.571.

    Laboratory method study · In six healthy volunteers, 60 minutes of treadmill running at 80 percent of peak oxygen uptake increased small intestinal permeability (lactulose-rhamnose) compared with rest, 40 and 60 percent, without more gastrointestinal symptoms.

  40. 40.

    Duerksen DR, Wilhelm-Boyles C, Veitch R, Kryszak D, Parry DM. A comparison of antibody testing, permeability testing, and zonulin levels with small-bowel biopsy in celiac disease patients on a gluten-free diet. Digestive Diseases and Sciences. 2010. PMID 19399613. DOI 10.1007/s10620-009-0813-5.

    Cross-sectional study · Of 21 celiac patients on a gluten-free diet for a mean 9.7 years, 10 had normalized biopsies, permeability and antibodies; 6 had Marsh 2 or 3 lesions, all with abnormal permeability (5 of 6) or TTG antibody (4 of 6); zonulin correlated with permeability only in Marsh 3 lesions.

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