title: Proteolysis is a confounding factor in the interpretation of faecal calprotectin.
abstract: BACKGROUND
  Calprotectin is a 36 kDa calcium and zinc binding protein. An increased level of calprotectin points towards inflammatory bowel disease. However, the biomarker calprotectin shows 14 potential cleavages sites for trypsin. Next to trypsin, also the presence of its inhibitor α1-antitrypsin after a gastrointestinal bleeding may affect calprotectin testing. In this study, effects of trypsin and α1-antitrypsin as potential confounders for faecal calprotectin testing are investigated.
  
  
  METHODS
  An in vitro model was created. As calprotectin source, leukocytes were isolated and subsequently lysed (1% Triton X-100) and diluted in faecal matrix. Trypsin digestion was carried out by adding trypsin. Incubation occurred for 24 h or 48 h (37 °C). To study the influence of α1-antitrypsin on trypsin, the same experiment was repeated after adding serum containing α1-antitrypsin.
  
  
  RESULTS
  In vitro experiments enabled monitoring of the faecal calprotectin digestion, leading to loss of immunoreactivity. Trypsin activity was a potential confounder in the interpretation of calprotectin, in particular for proximal lesions, where exposure of calprotectin to trypsin is prolonged. Relative calprotectin loss was proportional to the amount of trypsin. Decrease of calprotectin was more pronounced after 48 h of incubation in comparison to 24 h of incubation. Analogue experiments also showed stable calprotectin values after adding α1-antitrypsin.
  
  
  CONCLUSIONS
  Transit time, trypsin activity and addition of blood as a source of α1-antitrypsin may be regarded as potential confounders in the interpretation of calprotectin results. Age-related cut-off values depending on the anatomical localisation of the lesions could improve the diagnostic efficiency of calprotectin testing.
authors.forename: Els N
authors.initials: EN
authors.lastname: Dumoulin
authors.forename: Stephanie
authors.initials: S
authors.lastname: Van Biervliet
authors.forename: Michel R
authors.initials: MR
authors.lastname: Langlois
authors.forename: Joris R
authors.initials: JR
authors.lastname: Delanghe
mesh_terms: D000595:Amino Acid Sequence; D016477:Artifacts; D019963:Clinical Chemistry Tests; D005243:Feces / Q000737:chemistry*; D006801:Humans; D039841:Leukocyte L1 Antigen Complex / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D059748:Proteolysis; D014357:Trypsin / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism
chemical_list: D039841:Leukocyte L1 Antigen Complex; D000515:alpha 1-Antitrypsin; D014357:Trypsin