Measuring Myeloperoxidase Activity in Biological Samples

Abstract

BackgroundEnzymatic activity measurements of the highly oxidative enzyme myeloperoxidase (MPO), which is implicated in many diseases, are widely used in the literature, but often suffer from nonspecificity and lack of uniformity. Thus, validation and standardization are needed to establish a robust method that is highly specific, sensitive, and reproducible for assaying MPO activity in biological samples.Principal findingsWe found conflicting results between in vivo molecular MR imaging of MPO, which measures extracellular activity, and commonly used in vitro MPO activity assays. Thus, we established and validated a protocol to obtain extra- and intracellular MPO from murine organs. To validate the MPO activity assays, three different classes of MPO activity assays were used in spike and recovery experiments. However, these assay methods yielded inconsistent results, likely because of interfering substances and other peroxidases present in tissue extracts. To circumvent this, we first captured MPO with an antibody. The MPO activity of the resultant samples was assessed by ADHP and validated against samples from MPO-knockout mice in murine disease models of multiple sclerosis, steatohepatitis, and myocardial infarction. We found the measurements performed using this protocol to be highly specific and reproducible, and when performed using ADHP, to be highly sensitive over a broad range. In addition, we found that intracellular MPO activity correlated well with tissue neutrophil content, and can be used as a marker to assess neutrophil infiltration in the tissue.ConclusionWe validated a highly specific and sensitive assay protocol that should be used as the standard method for all MPO activity assays in biological samples. We also established a method to obtain extra- and intracellular MPO from murine organs. Extracellular MPO activity gives an estimate of the oxidative stress in inflammatory diseases, while intracellular MPO activity correlates well with tissue neutrophil content. A detailed step-by-step protocol is provided.

The Beneficial Effects of Hypiran in Ameliorating Rat Model of Ulcerative Colitis

“…It has been shown that hyperforin, a major component of the H. perforatum can inhibit COX-1 around 3-18 times similar to aspirin [37]. On the other hand, it has been documented that the COX-2 activity and production of inflammatory mediators by lipopolysaccharide-activated macrophage can be decreased by an ethanolic extract of H. perforatum or 4 compounds of this extract (pseudohypericin, amentoflavone, quercetin, and chlorogenic acid) [25]. It is proposed that the decrease in the activation of suppressor of cytokine signaling 3 protein (SOCS3) may partially participate in the reduction of COX-2 activity [38].…”

“…Myeloperoxidase, a peroxidase enzyme, is most abundantly found in neutrophil granulocytes and produces hypohalous acids during the neutrophil's respiratory burst and reactive oxygen production [25]. The excessive uncontrolled or inappropriate production of radical species may participate in the potentiation and prolongation of inflammatory injuries [41].…”

The roles of myeloperoxidase in coronary artery disease and its potential implication in plaque rupture

“…blood, plasma or tissue/cell lysates) prior to activity measurement has been utilized using different technologies [1,146] For example, Franck et al [147] developed the 'Specific Immunological Extraction followed by Enzymatic Detection' (SIEFED) method to detect MPO activity in human plasma and carotid extracts with a limit of detection of 0.08 mU/mL. The assay essentially immobilizes MPO onto a solid surface using anti-MPO antibodies, followed by addition of Amplex® Red (also known as ADHP) in the presence of H 2 O 2 and nitrite to detect MPO's peroxidase activity.…”

“…This assay has been applied to human plasma after stimulation of neutrophils in whole blood [148]. Others have utilized similar antibody-capture techniques of MPO from plasma or isolated leukocytes followed by detection of chlorinating activity with APF as a substrate for HOCl [146,149].…”

Synergistic effects of combined curcumin and antibiotic in ameliorating an animal model of Helicobacter pylori infection

“…Samples were incubated at 37°C for 5 min. Next, for termination of the reaction, 50 µl of 2 M H 2 SO 4 was added and the absorbance was determined spectrophotometrically at 450 nm [15].…”