MPO Activity Assay

Myeloperoxidase (MPO) activity was measured in colonic tissue as a parameter for tissue inflammation. The MPO activity directly correlates to the number of active neutrophil granulocytes as MPO is stored in the granules within the neutrophils and released upon degranulation. The assay was performed as described previously (Moreels et al., 2004; Ruyssers et al., 2009; Heylen et al., 2013). Briefly, colonic samples were taken in a standardized way, blotted dry, weighed and placed in a potassium phosphate buffer (pH = 6.0) containing 0.5% hexadecyltrimethylammoniumbromide at a ratio of 100 ml per 5 g tissue. Samples were homogenized and subjected to two freeze-thawing cycles. Subsequently, samples were centrifuged at 15,000 rpm for 15 min at 4°C. An aliquot (0.1 ml) of the supernatant was added to 2.9 ml of o-dianisidine solution (16.7 mg of o-dianisidine dihydrochloride in 1 ml of methyl alcohol, 98 ml of 50 mM potassium phosphate buffer at pH 6.0 and 1 ml of 0.005% H₂O₂ solution). The change in absorbance of the samples was read at 460 nm over 60 s using a Spectronic Genesys 5 spectrophotometer (Milton Roy). One unit of MPO activity equals the amount of enzyme able to convert 1 mmol of H₂O₂ to H₂O per minute at 25°C.