Measuring Myeloperoxidase Activity as a Marker of Inflammation in Gut Tissue Samples of Mice and Rat

Background

The presence of inflammatory infiltrates in the gastrointestinal tract is one of the hallmark characteristics of diseases such as ulcerative colitis and Crohn’s disease (Xavier and Podolsky, 2007). The pathogenesis of these diseases is frequently studied in rodents exposed to colitis-inducing chemicals such as acetic acid, dextran sodium sulfate (DSS), and 2,4,6-trinitrobenzene sulfonic acid (TNBS), or in genetically engineered mice that are prone to develop gastrointestinal inflammation spontaneously (Mizoguchi, 2012). To assess which factors contribute to the development, maintenance, and resolution of inflammation in the gut, the availability of sensitive assays to quantify this inflammation is essential.

Several methods are used to quantify the presence of inflammatory infiltrates in the gut. First, the degree of inflammation can be scored by quantifying the shortening and thickening of the intestinal wall via the weight:length ratio of the relevant region within the gastrointestinal tract (Solomon et al., 2010). Second, scoring systems can be used to assess the presence of ulcerations at an endoscopic, macroscopic, or microscopic level (Solomon et al., 2010; Heylen et al., 2013; Erben et al., 2014). Third, quantitative polymerase chain reactions (qPCR), bead-based immunoassays, or conventional enzyme-linked immunoassays (ELISAs) can be used to quantify the expression of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α) or interleukin 1β (IL-1β) (Khan et al., 2004; Yan et al., 2009; Kim et al., 2012). Finally, myeloperoxidase (MPO) activity can be measured as a biomarker of inflammation. MPO is a peroxidase contained within lysosomal azurophilic granules in neutrophils and released upon activation of the immune system (Schultz and Kaminker, 1962). It has been shown to correlate well with the number of neutrophils in histological sections and is therefore a simple, objective biochemical alternative to the labor-intensive histological analysis of inflammatory infiltrates (Krawisz et al., 1984).

The presence of MPO in tissue samples can be assessed in different ways. First, expression at the mRNA or protein level can be measured by means of qPCR or commercially available sandwich ELISAs, respectively. Second, kinetic assays can be used to evaluate enzymatic activity. In this protocol, we describe a simple, cost-effective kinetic colorimetric assay to determine MPO activity in intestinal tissue supernatant of mice. In addition, we compare our assay to an ELISA, demonstrating that the MPO concentration of the tissue does not necessarily reflect its enzymatic activity. The protocol consists of several steps. First, obtained tissue samples are homogenized in a potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB), which solubilizes the enzyme. Next, the supernatant is added to a reagent solution containing o-dianisidine dihydrochloride. This peroxidase substrate turns brownish upon oxidation and can be detected via spectrophotometry (λ = 460 nm).

This protocol was developed and used to measure MPO activity in intestinal tissues of mice and rat (Moreels et al., 2001; Ruyssers et al., 2009; Vermeulen et al., 2011). It could, however, be easily adapted to measure MPO activity in human gastrointestinal biopsies, or in samples derived from other organs. However, spectrophotometric interference by tissue myoglobin or vascular hemoglobin can occur, requiring adaptations of the protocol for organs such as the heart or brain (Kuebler et al., 1996; Xia and Zweier, 1997).