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Alessio Fasano - One of the best experts on this subject based on the ideXlab platform.
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Zonulin measurement conundrum add confusion to confusion does not lead to clarity
Gut, 2021Co-Authors: Alessio FasanoAbstract:I read with great interest the commentary by Massier et al 1 to the paper published in GUT by Talley et al 2 on their report suggesting that Zonulin is not a good gut permeability biomarker for irritable bowel disease, functional dyspepsia and non-coeliac gluten sensitivity. While I appreciate the authors’ effort in making the point of the poor reliability of the commercially available ELISA in measuring Zonulin, I think that some issues need to be clarified to avoid misleading the readership and, therefore, add more confusion to a field that would strongly benefit from greater clarity on this topic. Having been responsible for the discovery of Zonulin and for the development of the first ‘in house’ ELISA for measuring Zonulin, I think I am well positioned to provide the following clarifying points Since our discovery in 2000, …
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The therapeutic use of the Zonulin inhibitor AT-1001 (Larazotide) for a variety of acute and chronic inflammatory diseases.
Current medicinal chemistry, 2021Co-Authors: Jacopo Troisi, Giorgia Venutolo, Concetta Terracciano, Matteo Delli Carri, Simone Di Micco, Annamaria Landolfi, Alessio FasanoAbstract:Background The involvement of intercellular tight junctions and, in particular, the modulation of their competency by the Zonulin pathway with a subsequent increase in epithelial and endothelial permeability, has been described in several chronic and acute inflammatory diseases. In this scenario, Larazotide, a Zonulin antagonist, could be employed as a viable therapeutic strategy. Objective The present review aims to describe recent research and current observations about Zonulin involvement in several diseases and the use of its inhibitor Larazotide for their treatment. Methods A systematic search was conducted on PubMed and Google Scholar, resulting in 209 publications obtained with the following search query: "Larazotide," "Larazotide acetate," "AT-1001," "FZI/0" and "INN-202." After careful examination, some publications were removed from consideration because they were either not in English or were not directly related to Larazotide. Results The obtained publications were subdivided according to Larazotide's mechanism of action and different diseases: celiac disease, type 1 diabetes, other autoimmune diseases, inflammatory bowel disease, Kawasaki disease, respiratory (infective and/or non-infective) diseases, and other. Conclusions A substantial role of Zonulin in many chronic and acute inflammatory diseases has been demonstrated in both in vivo and in vitro, indicating the possible efficacy of a Larazotide treatment. Moreover, new possible molecular targets for this molecule have also been demonstrated.
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Zonulin dependent intestinal permeability in children diagnosed with mental disorders a systematic review and meta analysis
Nutrients, 2020Co-Authors: Birna Guðrun Asbjornsdottir, Alessio Fasano, Heiðdis Snorradottir, Edda Andresdottir, Bertrand Lauth, Larus S Gudmundsson, Magnus Gottfredsson, Thorhallur I Halldorsson, Bryndis Eva BirgisdottirAbstract:Worldwide, up to 20% of children and adolescents experience mental disorders, which are the leading cause of disability in young people. Research shows that serum Zonulin levels are associated with increased intestinal permeability (IP), affecting neural, hormonal, and immunological pathways. This systematic review and meta-analysis aimed to summarize evidence from observational studies on IP in children diagnosed with mental disorders. The review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A systematic search of the Cochrane Library, PsycINFO, PubMed, and the Web of Science identified 833 records. Only non-intervention (i.e., observational) studies in children (<18 years) diagnosed with mental disorders, including a relevant marker of intestinal permeability, were included. Five studies were selected, with the risk of bias assessed according to the Newcastle–Ottawa scale (NOS). Four articles were identified as strong and one as moderate, representing altogether 402 participants providing evidence on IP in children diagnosed with attention deficit and hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and obsessive–compulsive disorder (OCD). In ADHD, elevated serum Zonulin levels were associated with impaired social functioning compared to controls. Children with ASD may be predisposed to impair intestinal barrier function, which may contribute to their symptoms and clinical outcome compared to controls. Children with ASD, who experience gastro-intestinal (GI) symptoms, seem to have an imbalance in their immune response. However, in children with OCD, serum Zonulin levels were not significantly different compared to controls, but serum claudin-5, a transmembrane tight-junction protein, was significantly higher. A meta-analysis of mean Zonulin plasma levels of patients and control groups revealed a significant difference between groups (p = 0.001), including the four studies evaluating the full spectrum of the Zonulin peptide family. Therefore, further studies are required to better understand the complex role of barrier function, i.e., intestinal and blood–brain barrier, and of inflammation, to the pathophysiology in mental and neurodevelopmental disorders. This review was PROSPERO preregistered, (162208).
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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, 2018Co-Authors: Lucas Scheffler, Alyce Crane, Henrike O Heyne, Anke Tonjes, Dorit Schleinitz, Christian Ihling, Michael Stumvoll, Rachel Freire, Maria Fiorentino, Alessio FasanoAbstract:BACKGROUND. There is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating Zonulin and metabolic traits related to obesity. METHODS. Serum Zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects. RESULTS. As Zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the Zonulin ELISA assay using antibody capture experiments, mass spectrometry and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin 2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against Zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the Zonulin family. CONCLUSIONS. Our study suggests that the Zonulin ELISA does not recognize pre-haptoglobin 2, rather structural (and possibly functional) analogue proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.
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widely used commercial elisa does not detect precursor of haptoglobin2 but recognizes properdin as a potential second member of the Zonulin family
bioRxiv, 2018Co-Authors: Lucas Scheffler, Alyce Crane, Henrike O Heyne, Anke Tonjes, Dorit Schleinitz, Christian Ihling, Michael Stumvoll, Rachel Freire, Maria Fiorentino, Alessio FasanoAbstract:Background: There is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating Zonulin and metabolic traits related to obesity. Methods: Serum Zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects. Results: As Zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the Zonulin ELISA assay using antibody capture experiments, mass spectrometry, and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against Zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the Zonulin family. Conclusion: Our study suggests that the Zonulin ELISA does not recognize pre-haptoglobin2, rather structural (and possibly functional) analog proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.
Kirsi Laitinen - One of the best experts on this subject based on the ideXlab platform.
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increased intestinal permeability measured by serum Zonulin is associated with metabolic risk markers in overweight pregnant women
Metabolism-clinical and Experimental, 2017Co-Authors: Kati Mokkala, Henna Roytio, Tapani Ronnemaa, Outi Pellonpera, Pirkko J Pussinen, Kirsi LaitinenAbstract:Abstract Background Increased intestinal permeability with subsequent metabolic endotoxemia, i.e., elevated circulating levels of bacterial lipopolysaccharide, LPS, has been introduced as a novel initiator of obesity related metabolic disturbances in non-pregnant individuals. The objective was to investigate the extent to which intestinal permeability, measured by serum Zonulin concentration, is related to metabolic endotoxemia and metabolic risk markers in overweight pregnant women. Methods This was a cross-sectional study including 100 pregnant overweight women in early pregnancy. Serum Zonulin was analyzed using ELISA, and markers for metabolic endotoxemia (LPS), inflammation (high-sensitive C-reactive protein and glycoprotein acetylation GlyA), glucose metabolism (fasting glucose and insulin), and lipid metabolism were measured. Results Higher serum Zonulin concentration associated positively with LPS ( P =0.02), inflammatory markers ( P P P P =0.001), and negatively with insulin sensitivity ( P =0.001) (ANOVA with Tukey's corrections or Kruskal–Wallis nonparametric test with Bonferroni correction for Zonulin quartiles). All the observed associations were confirmed ( P Conclusions Our findings suggest that increased serum Zonulin concentration, i.e., increased intestinal permeability, contributes to metabolic endotoxemia, systemic inflammation, and insulin resistance in overweight pregnant women. By reinforcing intestinal barrier, it may be possible to manipulate maternal metabolism during pregnancy with subsequent health benefits.
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evaluation of serum Zonulin for use as an early predictor for gestational diabetes
Nutrition & Diabetes, 2017Co-Authors: Kati Mokkala, Tapani Ronnemaa, Kristiina Tertti, Tero Vahlberg, Kirsi LaitinenAbstract:Diet has an important role in regulating intestinal permeability and subsequently the risk for metabolic disorders. In this observational study, we examined whether serum intestinal permeability marker Zonulin, could be used as a predictor for gestational diabetes mellitus (GDM). Serum Zonulin concentration was measured in early pregnancy in overweight or obese pregnant women (n=88) at risk for developing GDM. Serum Zonulin was associated with higher odds of GDM (adjusted OR for 1 ng ml−1 increase in Zonulin: 1.08, 95% CI: 1.02–1.15; P=0.009), diagnosed by a 2-h 75-g oral glucose tolerance test at late pregnancy. The optimal cutoff value was 43.3 ng ml−1, with sensitivity of 88% (95% CI: 71–100%) and specificity of 47% (95% CI: 33–58%). The area under the ROC-curve was 0.67 (95% CI: 0.54–0.81). Our results show an association between increased early-pregnancy serum Zonulin concentration and GDM, suggesting Zonulin as a possible predictor for GDM.
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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, 2016Co-Authors: Kati Mokkala, Henna Roytio, Eveliina Munukka, Sami Pietila, Ulla Ekblad, Tapani Ronnemaa, Erkki Eerola, Asta Laiho, Kirsi LaitinenAbstract:BACKGROUND Increased intestinal permeability may precede adverse metabolic conditions. The extent to which the composition of the gut microbiota and diet contribute to intestinal permeability during pregnancy is unknown. OBJECTIVE The aim was to investigate whether the gut microbiota and diet differ according to serum Zonulin concentration, a marker of intestinal permeability, in overweight pregnant women. METHODS This cross-sectional study included 100 overweight women [mean age: 29 y; median body mass index (in kg/m(2)): 30] in early pregnancy (<17 wk of gestation; median: 13 wk). Serum Zonulin (primary outcome) was determined by using ELISA, gut microbiota by 16S ribosomal RNA sequencing, and dietary intake of macro- and micronutrients from 3-d food diaries. The Mann-Whitney U test was used for pairwise comparisons and linear regression and Spearman's nonparametric correlations for relations between serum Zonulin and other outcome variables. RESULTS Women were divided into "low" (<46.4 ng/mL) and "high" (≥46.4 ng/mL) serum Zonulin groups on the basis of the median concentration of Zonulin (46.4 ng/mL). The richness of the gut microbiota (Chao 1, observed species and phylogenetic diversity) was higher in the low Zonulin group than in the high Zonulin group (P = 0.01). The abundances of Bacteroidaceae and Veillonellaceae, Bacteroides and Blautia, and Blautia sp. were lower and of Faecalibacterium and Faecalibacterium prausnitzii higher (P < 0.05) in the low Zonulin group than in the high Zonulin group. Dietary quantitative intakes of n-3 (ω-3) polyunsaturated fatty acids (PUFAs), fiber, and a range of vitamins and minerals were higher (P < 0.05) in women in the low Zonulin group than those in the high Zonulin group. CONCLUSIONS The richness and composition of the gut microbiota and the intake of n-3 PUFAs, fiber, and a range of vitamins and minerals in overweight pregnant women are associated with serum Zonulin concentration. Modification of the gut microbiota and diet may beneficially affect intestinal permeability, leading to improved metabolic health of both the mother and fetus. This trial was registered at clinicaltrials.gov as NCT01922791.
Peter R. Gibson - One of the best experts on this subject based on the ideXlab platform.
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serum Zonulin as a marker of intestinal mucosal barrier function may not be what it seems
PLOS ONE, 2019Co-Authors: Mary Ajamian, David Steer, Gennaro Rosella, Peter R. GibsonAbstract:The protein, Zonulin, has emerged as a popular serological marker to assess the integrity of the intestinal mucosal barrier. However, there is limited information on the utility of serum Zonulin to indicate gastrointestinal disease and the validity of Zonulin detection in widely-used commercial assays. The current study reports differences in Zonulin levels across patient groups with gastrointestinal dysfunction compared with healthy individuals, though methodological inconsistencies indicated that actual Zonulin protein was not detected by the commercial assays applied. The nature of the assays' detected antigen was investigated using immunoprecipitation followed by mass spectrometric analysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein staining. Top matches of the assays' detected antigen included haptoglobin and complement C3 for the assay manufactured by CUSABIO (Wuhan, China) and complement C3 for the assay manufactured by Immundiagnostik AG (Bensheim, Germany). These findings confirm that current commercial Zonulin assays are not detecting the actual protein as prehaptoglobin-2. Until assay methodology is improved, we advise the greater scientific and medical community to exercise caution in considering the measurement of serum Zonulin as a marker of mucosal barrier integrity.
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Visualisation of immunoprecipitated protein products and standards.
2019Co-Authors: Mary Ajamian, David Steer, Gennaro Rosella, Peter R. GibsonAbstract:(A) 5 μg of human complement C3c, HP1-1, HP2-2 and recombinant Zonulin (zon) standards were separated by SDS-PAGE and stained with Coomassie gel stain. Characteristic bands of standards include the conserved C3c β-chain at 70 kDa, the haptoglobin β-chain at 40 kDa, and the 47 kDa band indicative of recombinant Zonulin. (B) Silver staining of immunoprecipitated protein samples detected by LC-MS/MS. Lanes (1) and (2) contain immunoprecipitated proteins from incubation with serum in commercial assays. Lane (1) contains serum proteins captured by CUSABIO assay, which includes complement C3 and haptoglobin as identified by mass spectrometry. Bands at 70 kDa, suggestive of complement C3, and haptoglobin at 40 kDa are present. Lane (2) contains serum proteins captured by Immundiagnostik assay, which includes complement C3 as identified by mass spectrometry. Lanes (3) and (4) contain immunoprecipitated product from incubation of commercial kits with recombinant Zonulin. Lane (3), which includes immunoprecipitated product captured by CUSABIO assay, contains trace amounts of protein around the 45–65 kDa range. Mass spectrometry results also indicated a small amount of haptoglobin (S1 File), which remained undetected by silver stain. Lane (4) contained immunoprecipitated product captured by Immundiagnostik assay; no significant detectable proteins aside from albumin were detected by mass spectrometry (S1 File) or indicated by silver stain.
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Purported serum Zonulin levels (ng/mL) in Zonulin producers detected by CUSABIO ELISA assay.
2019Co-Authors: Mary Ajamian, David Steer, Gennaro Rosella, Peter R. GibsonAbstract:Levels of Zonulin, as advertised, were determined in non-coeliac wheat sensitivity (NCWS) (n = 36), coeliac disease, (n = 37), and ulcerative colitis (n = 20) patients as well as healthy individuals (n = 49). Compared with the cohort of healthy individuals, patient Zonulin levels were elevated (all p
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Comparison of purported serum Zonulin levels (ng/mL) between CUSABIO and Immundiagnostik ELISA assays.
2019Co-Authors: Mary Ajamian, David Steer, Gennaro Rosella, Peter R. GibsonAbstract:Selected study samples (n = 28) ran in duplicate were compared. (A) Correlation between the two assays (p = 0.14, Spearman’s r = 0.29). (B) Bland-Altman plot calculating difference in Zonulin levels vs average of Zonulin levels (bias/average discrepancy between methods was -31.62 ng/mL, 95% limits of agreement were from -88.51 to 25.28 ng/mL).
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Identification of proteins immunoprecipitated from commercial Zonulin ELISA assays by LC-MS/MS.
2019Co-Authors: Mary Ajamian, David Steer, Gennaro Rosella, Peter R. GibsonAbstract:Identification of proteins immunoprecipitated from commercial Zonulin ELISA assays by LC-MS/MS.
Anette H.h. Van Boxel-dezaire - One of the best experts on this subject based on the ideXlab platform.
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IFN-γ, IL-17A, or Zonulin rapidly increase the permeability of the blood-brain and small intestinal epithelial barriers: Relevance for neuro-inflammatory diseases
Biochemical and Biophysical Research Communications, 2018Co-Authors: Mohammed Rahman, Chaitali Ghosh, Mohammed Hossain, Debra Linfield, Fariba Rezaee, Damir Janigro, Nicola Marchi, Anette H.h. Van Boxel-dezaireAbstract:Breakdown of the blood-brain barrier (BBB) precedes lesion formation in the brains of multiple sclerosis (MS) patients. Since recent data implicate disruption of the small intestinal epithelial barrier (IEB) in the pathogenesis of MS, we hypothesized that the increased permeability of the BBB and IEB are mechanistically linked. Zonulin, a protein produced by small intestine epithelium, can rapidly increase small intestinal permeability. Zonulin blood levels are elevated in MS, but it is unknown whether Zonulin can also disrupt the BBB. Increased production of IL-17A and IFN-γ has been implicated in the pathogenesis of MS, epilepsy, and stroke, and these cytokines impact BBB integrity after 24 h. We here report that primary human brain microvascular endothelial cells expressed the EGFR and PAR2 receptors necessary to respond to Zonulin, and that Zonulin increased BBB permeability to a 40 kDa dextran tracer within 1 h. Moreover, both IL-17A and IFN-γ also rapidly increased BBB and IEB permeability. By using confocal microscopy, we found that exposure of the IEB to Zonulin, IFN-γ, or IL-17A in vitro rapidly modified the localization of the TJ proteins, ZO-1, claudin-5, and occludin. TJ disassembly was accompanied by marked depolymerization of the peri-junctional F-actin cytoskeleton. Our data indicate that IFN-γ, IL-17A, or Zonulin can increase the permeability of the IEB and BBB rapidly in vitro, by modifying TJs and the underlying actin cytoskeleton. These observations may help clarify how the gut-brain axis mediates the pathogenesis of neuro-inflammatory diseases.
Kati Mokkala - One of the best experts on this subject based on the ideXlab platform.
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increased intestinal permeability measured by serum Zonulin is associated with metabolic risk markers in overweight pregnant women
Metabolism-clinical and Experimental, 2017Co-Authors: Kati Mokkala, Henna Roytio, Tapani Ronnemaa, Outi Pellonpera, Pirkko J Pussinen, Kirsi LaitinenAbstract:Abstract Background Increased intestinal permeability with subsequent metabolic endotoxemia, i.e., elevated circulating levels of bacterial lipopolysaccharide, LPS, has been introduced as a novel initiator of obesity related metabolic disturbances in non-pregnant individuals. The objective was to investigate the extent to which intestinal permeability, measured by serum Zonulin concentration, is related to metabolic endotoxemia and metabolic risk markers in overweight pregnant women. Methods This was a cross-sectional study including 100 pregnant overweight women in early pregnancy. Serum Zonulin was analyzed using ELISA, and markers for metabolic endotoxemia (LPS), inflammation (high-sensitive C-reactive protein and glycoprotein acetylation GlyA), glucose metabolism (fasting glucose and insulin), and lipid metabolism were measured. Results Higher serum Zonulin concentration associated positively with LPS ( P =0.02), inflammatory markers ( P P P P =0.001), and negatively with insulin sensitivity ( P =0.001) (ANOVA with Tukey's corrections or Kruskal–Wallis nonparametric test with Bonferroni correction for Zonulin quartiles). All the observed associations were confirmed ( P Conclusions Our findings suggest that increased serum Zonulin concentration, i.e., increased intestinal permeability, contributes to metabolic endotoxemia, systemic inflammation, and insulin resistance in overweight pregnant women. By reinforcing intestinal barrier, it may be possible to manipulate maternal metabolism during pregnancy with subsequent health benefits.
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evaluation of serum Zonulin for use as an early predictor for gestational diabetes
Nutrition & Diabetes, 2017Co-Authors: Kati Mokkala, Tapani Ronnemaa, Kristiina Tertti, Tero Vahlberg, Kirsi LaitinenAbstract:Diet has an important role in regulating intestinal permeability and subsequently the risk for metabolic disorders. In this observational study, we examined whether serum intestinal permeability marker Zonulin, could be used as a predictor for gestational diabetes mellitus (GDM). Serum Zonulin concentration was measured in early pregnancy in overweight or obese pregnant women (n=88) at risk for developing GDM. Serum Zonulin was associated with higher odds of GDM (adjusted OR for 1 ng ml−1 increase in Zonulin: 1.08, 95% CI: 1.02–1.15; P=0.009), diagnosed by a 2-h 75-g oral glucose tolerance test at late pregnancy. The optimal cutoff value was 43.3 ng ml−1, with sensitivity of 88% (95% CI: 71–100%) and specificity of 47% (95% CI: 33–58%). The area under the ROC-curve was 0.67 (95% CI: 0.54–0.81). Our results show an association between increased early-pregnancy serum Zonulin concentration and GDM, suggesting Zonulin as a possible predictor for GDM.
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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, 2016Co-Authors: Kati Mokkala, Henna Roytio, Eveliina Munukka, Sami Pietila, Ulla Ekblad, Tapani Ronnemaa, Erkki Eerola, Asta Laiho, Kirsi LaitinenAbstract:BACKGROUND Increased intestinal permeability may precede adverse metabolic conditions. The extent to which the composition of the gut microbiota and diet contribute to intestinal permeability during pregnancy is unknown. OBJECTIVE The aim was to investigate whether the gut microbiota and diet differ according to serum Zonulin concentration, a marker of intestinal permeability, in overweight pregnant women. METHODS This cross-sectional study included 100 overweight women [mean age: 29 y; median body mass index (in kg/m(2)): 30] in early pregnancy (<17 wk of gestation; median: 13 wk). Serum Zonulin (primary outcome) was determined by using ELISA, gut microbiota by 16S ribosomal RNA sequencing, and dietary intake of macro- and micronutrients from 3-d food diaries. The Mann-Whitney U test was used for pairwise comparisons and linear regression and Spearman's nonparametric correlations for relations between serum Zonulin and other outcome variables. RESULTS Women were divided into "low" (<46.4 ng/mL) and "high" (≥46.4 ng/mL) serum Zonulin groups on the basis of the median concentration of Zonulin (46.4 ng/mL). The richness of the gut microbiota (Chao 1, observed species and phylogenetic diversity) was higher in the low Zonulin group than in the high Zonulin group (P = 0.01). The abundances of Bacteroidaceae and Veillonellaceae, Bacteroides and Blautia, and Blautia sp. were lower and of Faecalibacterium and Faecalibacterium prausnitzii higher (P < 0.05) in the low Zonulin group than in the high Zonulin group. Dietary quantitative intakes of n-3 (ω-3) polyunsaturated fatty acids (PUFAs), fiber, and a range of vitamins and minerals were higher (P < 0.05) in women in the low Zonulin group than those in the high Zonulin group. CONCLUSIONS The richness and composition of the gut microbiota and the intake of n-3 PUFAs, fiber, and a range of vitamins and minerals in overweight pregnant women are associated with serum Zonulin concentration. Modification of the gut microbiota and diet may beneficially affect intestinal permeability, leading to improved metabolic health of both the mother and fetus. This trial was registered at clinicaltrials.gov as NCT01922791.