A spectrophotometric assay for tissue-associated myeloperoxidase activity and its application to intestinal ischemia-reperfusion

Abstract

Acute inflammation is manifested by polymorphonuclear leukocyte infiltration into the affected tissue. Myeloperoxidase (MPO) activity can be used to monitor this stiuation, since this enzyme is involved in the microbicidal activity of neutrophils and thus it is a significant biochemical marker for their presence. We optimized a spectrophotometric method to assay tissue MPO activity. This method uses 3,3', 5,5' - tetramethyl benzydine (TMB) as an oxidizable dye, and the reaction is started by adding hydrogen peroxide (H2O2) in the medium. The assay is based on the oxidation of TMB by MPO in the presence of H2O2. In assay conditions, we used TMB at 1.6 mM and H2O2 at 0.88 mM. At these concentrations, neither substrates were rate-limiting. Acute inflammatory response is usually high in the small intestine, and this tissue is also very sensitive to ischemia-reperfusion injury. In the second part of our study, we looked for MPO activity in the ratÕs small intestine after various periods of ischemia and reperfusion. We showed that this method could be successfully used to monitor ischemia-reperfusion injury of this organ, and we discovered that in order to observe maximal reperfusion injury, it was essential to restrict the ischemic period to 30 minutes. Beyond this time, ischemic injury dominated and overwhelmed the reperfusion component.