What is a Zonulin Test and Its Indications
Commercially available zonulin tests should not be used in clinical practice because they do not measure actual zonulin (pre-haptoglobin-2) but instead detect complement proteins, rendering the results clinically meaningless. 1, 2
Understanding Zonulin
Zonulin is a physiological modulator of intestinal tight junctions, identified as the precursor form of haptoglobin-2 (pre-HP2). 3 In its uncleaved precursor form, zonulin regulates intestinal permeability by activating the EGF receptor via proteinase-activated receptor 2 (PAR2), leading to reversible opening of tight junctions. 3 Once cleaved into its mature α2- and β-subunits (forming haptoglobin), it loses this permeability-modulating function and instead scavenges free hemoglobin. 3
Critical Problem with Commercial Testing
The fundamental issue is that widely-used commercial ELISA assays (CUSABIO and Immundiagnostik AG) do not detect true zonulin. 1, 2 Mass spectrometric analysis and immunoprecipitation studies revealed these assays actually detect:
- Haptoglobin and complement C3 proteins (CUSABIO assay) 2
- Complement C3 proteins (Immundiagnostik AG assay) 2
This methodological flaw invalidates all research and clinical conclusions based on these commercial assays. 1 Studies claiming benefits of interventions on "zonulin levels" using these tests are unreliable because they measured the wrong proteins entirely. 1
Theoretical Indications (If Valid Testing Were Available)
Research suggests zonulin measurement could theoretically be useful for:
- Celiac disease assessment: Zonulin expression is elevated in intestinal mucosa during acute celiac disease, correlating with increased intestinal permeability. 3, 4
- Non-celiac gluten sensitivity (NCGS): One study using serum zonulin reported 81% diagnostic accuracy in distinguishing NCGS from IBS-D, improving to 89% when combined with symptoms and gender. 5 However, given the assay validity concerns, these findings require confirmation with accurate zonulin measurement methods.
- Metabolic dysfunction-associated steatotic liver disease (MASLD): Fecal zonulin showed modest association with hepatic steatosis (AUC 0.680), though the clinical utility remains limited. 6
Clinical Recommendations
Do not order commercial zonulin tests for any clinical indication. 1 The major pitfall is relying on these tests for clinical decision-making about intestinal permeability, leaky gut syndrome, or inflammatory conditions. 1, 2
Important distinction: Zonulin (pre-haptoglobin-2) is completely different from Zonula occludens proteins (ZO-1, ZO-2, ZO-3), which are structural tight junction proteins studied in barrier research but not measured by commercial zonulin assays. 1
Until assay methodology is corrected to specifically detect pre-haptoglobin-2, serum or fecal zonulin measurement has no valid role in clinical practice. 2 Any systematic review that included zonulin measurements using these flawed assays appropriately excluded such studies from analysis due to invalid methodology. 1