Measuring Myeloperoxidase Activity in Biological Samples

Background: Enzymatic 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 findings We 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. Conclusion: We 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

Regionally Metastatic Merkel Cell Carcinoma Associated with Paraneoplastic Anti-N-methyl-D-aspartate Receptor Encephalitis

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous neuroendocrine cancer with a high risk of recurrence and metastasis. MCC is generally associated with advanced age, fair skin, sun exposure, immunosuppression, and in the majority of cases, the Merkel cell polyomavirus. Neuroendocrine malignancies are associated with a variety of paraneoplastic neurological syndromes (PNS), characterized as autoimmune responses to malignancy-associated expression of neural antigens. Our literature review underscores previous case reports of MCC-associated PNS with voltage-gated calcium channel (VGCC) and anti-Hu (or ANNA-1) autoantibodies. We present the case of a 59-year-old male with regionally metastatic Merkel cell carcinoma complicated by the paraneoplastic manifestation of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. His primary lower neck subcutaneous MCC and metastasis were initially treated with surgery. Additional recurrent lymph node metastases were successfully treated with definitive intensity-modulated radiation therapy. His PNS improved with rituximab therapy. Although rare, this case highlights that in the setting of seizures and prominent psychiatric symptoms accompanying an MCC diagnosis, evaluation for autoimmune paraneoplastic encephalitis is warranted. Awareness and detection of preexisting PNS are crucial in the era of immune checkpoint inhibitors (ICI) for advanced MCC, where treatment with ICI has the potential to exacerbate preexisting autoimmune PNS and lead to worsened or even lethal neurologic immune-related adverse events (nirAEs)

<i>In vivo</i> imaging and <i>in vitro</i> MPO activity assays demonstrate markedly different findings.

<p>(<b>A</b>) MPO-Gd molecular MR imaging reveals MPO inhibition in vivo in mice with experimental autoimmune encephalomyelitis that were treated with ABAH. MPO activity maps are shown in 3D from two angles (left), as well as overlays of MPO activity maps over T1 images (right). (<b>B</b>) Quantification of imaging reveals significant difference in MPO activity <i>in vivo</i> (<i>P</i> = 0.03, n = 8 per group). (<b>C</b>) <i>In vitro</i> assays on whole tissue homogenates using ADHP or TMB do not confirm the <i>in vivo</i> imaging finding (<i>P</i> = 0.68 and 0.88, respectively, n = 4 per group). *: <i>P</i><0.05, n.s. = not statistically significant. MPO = myeloperoxidase. TMB = 3,3′,5,5′-Tetramethylbenzidine. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. ABAH = 4-aminobenzoic acid hydrazide. Activation ratio = contrast-to-noise ratio 60 minutes over 15 minutes post MPO-Gd injection.</p

Intracellular MPO activity correlates well with tissue neutrophil content.

<p>(<b>A</b>) Flow cytometry demonstrates different neutrophil counts in brain, heart, liver, spleen, and bone marrow, as quantified in (<b>B</b>) (n = 2 per group). (<b>C</b>) Intracellular MPO activity was measured with the antibody-capture assay using ADHP, and shows a similar trend to neutrophil content per organ (n = 2 per group). (<b>D</b>) A close correlation was found between neutrophil content and intracellular MPO activity in these organs. MPO = myeloperoxidase. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine.</p

Antibody capture improves the specificity of MPO activity assays on extra- and intracellular extracts in various models of inflammatory diseases.

<p>(<b>A</b>) Antibody capture of MPO followed by activity detection with ADHP reveals high specificity towards MPO. This is shown in extra- and intracellular fractions in brains from EAE mice, livers from mice with NASH, and hearts and plasma from mice with myocardial infarction (n = 3 per group). (<b>B</b>) The same samples processed without antibody capture reveal poor specificity towards MPO, and no significant difference between WT and MPO-KO mice (n = 3 per group). * <i>P</i><0.05. ** <i>P</i><0.01. *** <i>P</i><0.001. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. MPO = myeloperoxidase. EAE = experimental autoimmune encephalomyelitis. MI = myocardial infarction. NASH = non-alcoholic steatohepatitis. ECF = extracellular fraction. ICF = intracellular fraction. WT = wildtype C57BL/6. MPO^−/− = MPO knockout.</p

Spike and recovery assay: tissue homogenates and extracellular fluid contain interfering substances.

<p>(<b>A</b>) Extracellular protein fraction from different organs contains substances interfering with ADHP, luminol, and APF assays (n = 2 per group). (<b>B</b>) Intracellular protein fractions also contain interfering substances (n = 2 per group). MPO = myeloperoxidase. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. APF = 3′-(p-aminophenyl) fluorescein. HPF = 3′-(p-hydroxyphenyl) fluorescein.</p

Validation of Extracellular Protein Isolation and MPO Protein Precipitation.

<p>(<b>A</b>) LDH assay of intra- and extracellular protein fractions of different organs shows that the extracellular fraction only contains very low levels of LDH activity, while the intracellular fraction contains the majority of the LDH activity (left). LDH ratio shows a 90 or higher fold level of ICF LDH over ECF LDH activity (right). (<b>B</b>) Protein precipitation of MPO with acetone has no effect on its activity, as evaluated with ADHP (n = 2 per group). LDH = lactate dehydrogenase. BCA = bicinchoninic acid. MPO = myeloperoxidase.</p

MPO in the literature.

<p>(<b>A</b>) Usage of MPO activity assays in the Literature from 2011 to 2012. (<b>B</b>) Manuscripts published on MPO from 1990 to 2012; grey bars indicate manuscripts considered in (<b>A</b>). MPO = myeloperoxidase. TMB = 3,3′,5,5′-Tetramethylbenzidine. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. BALF = bronchoalveolar lavage fluid. Ab = antibody. APF = 3′-(p-aminophenyl) fluorescein. ELISA = enzyme-linked immunosorbent assay.</p