BackgroundThere 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.MethodsSerum 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.ResultsAs 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.ConclusionOur 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.

Keywords: diabetes -lipid metabolism -pre-Haptoglobin 2 -ELISA -intestinal permeability 25 -properdin -obesity -metabolic diseases 26 27 Nonstandard abbreviations: HP, haptoglobin; T2D, type 2 diabetes; WHR, waist-to-hip 28 ratio.Abstract 30 BACKGROUND. There is increasing evidence for the role of impaired intestinal 31 permeability in obesity and associated metabolic diseases. Zonulin is an established serum 32 marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to 33 investigate the relationship between circulating zonulin and metabolic traits related to obesity. 34METHODS. Serum zonulin was measured by using a widely used commercial ELISA kit in 35 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In 36 addition, haptoglobin genotype was determined in DNA samples from all study subjects. 37 RESULTS.As zonulin concentrations did not correlate to the haptoglobin genotypes, we 38 investigated the specificity of the zonulin ELISA assay using antibody capture experiments, 39 mass spectrometry and Western blot analysis. Using serum samples that gave the highest or 40 lowest ELISA signals, we detected several proteins that are likely to be captured by the 41 antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. 42 We used increasing concentrations of recombinant pre-haptoglobin 2 and complement C3 as 43 one of the representative captured proteins and the ELISA kit did not detect either. Western 44 blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies 45 rose against zonulin showed a similar protein recognition pattern but with different intensity 46 of detection. The protein(s) measured using the ELISA kit was (were) significantly increased 47 in patients with diabetes and obesity and correlated strongly with markers of the lipid and 48 glucose metabolism. Combining mass spectrometry and Western blot analysis using the 49 polyclonal antibodies used in the ELISA kit, we identified properdin as another member of 50 the zonulin family. 51 CONCLUSIONS. Our study suggests that the zonulin ELISA does not recognize pre-52 haptoglobin 2, rather structural (and possibly functional) analogue proteins belonging to the 53 mannose-associated serine protease family, with properdin being the most likely possible 54 candidate. 55 56 The "intestinal barrier" is an established term, defined as a functional entity separating the gut 57 lumen from the inner host, and consisting of mechanical, humoral, immunological, muscular 58 and neurological elements. Intestinal barrier dysfunction is a characteristic feature of 59 pathological states such as inflammatory bowel disease, celiac disease, nonalcoholic 60 steatohepatitis and ulcerative colitis (1, 2). There is also emerging evidence for the role of 61 impaired intestinal permeability in metabolic diseases including obesity and type 2 diabetes 62 (T2D) (3-5). It has been hypothesized that gut bacteria and bacterial endotoxins may disrup...