Department of Biomedical and Specialist Surgical Sciences, Section of Medical Biochemistry, Molecular Biology and Genetics, University of Ferrara, Ferrara, Italy.
Department of Morphology, Surgery and Experimental Medicine, Section of Anesthesia and Intensive Care University of Ferrara, Ferrara, Italy.
Clin Chem Lab Med. 2020 Sep 25;58(10):1749-1758. doi: 10.1515/cclm-2019-0817.
Background Myeloperoxidase (MPO) is an enzyme with a recognized prognostic role in coronary artery disease (CAD), which is also emerging as a promising biomarker for cardiac risk stratification. However, the lack of a consensus method for its quantification has hindered its implementation in clinical practice. The aim of our work was to optimize an absolute sensitive assay for active MPO without external standards, to validate the method in the clinical context of CAD patients, and to estimate the enzyme specific activity. Methods In order to determine the MPO concentration using fluorescence readings, this ELISA assay exploits the activity of the enzyme recognized by specific antibodies. The assay was validated in a small cohort of patients that included: healthy subjects (n=60); patients with acute myocardial infarction (AMI, n=25); patients with stable CAD (SCAD, n=25) and a concomitant chronic obstructive pulmonary disease (COPD). Then, total MPO concentration and specific activity (activity/total MPO) were determined. Results The assay showed an intra- and inter-assay coefficient of variation of 5.8% and 10.4%, respectively, with a limit of detection (LoD) of 0.074 μU. Both AMI and SCAD patients had higher active and total MPO than controls (p<0.0001 and p<0.01, respectively). The specific activity of MPO was higher in SCAD patients compared to both controls and AMI (p<0.0001). Conclusions The study presents a robust and sensitive method for assaying MPO activity in biological fluids with low variability. Moreover, the determination of the specific activity could provide novel insight into the role of MPO in cardiovascular diseases (CVDs).
髓过氧化物酶（MPO）是一种在冠状动脉疾病（CAD）中具有公认预后作用的酶，它也正在成为心脏风险分层的有前途的生物标志物。然而，缺乏用于其定量的共识方法阻碍了其在临床实践中的应用。我们的工作旨在优化一种无外部标准的活性 MPO 的绝对敏感测定法，在 CAD 患者的临床背景下验证该方法，并估计酶的比活性。
为了使用荧光读数来确定 MPO 浓度，该 ELISA 测定法利用了被特定抗体识别的酶的活性。该测定法在一个包括以下患者的小队列中得到了验证：健康受试者（n=60）；急性心肌梗死（AMI，n=25）患者；稳定性 CAD（SCAD，n=25）患者和同时患有慢性阻塞性肺疾病（COPD）的患者。然后，确定了总 MPO 浓度和比活性（活性/总 MPO）。
该测定法的日内和日间变异系数分别为 5.8%和 10.4%，检测限（LoD）为 0.074 μU。AMI 和 SCAD 患者的活性和总 MPO 均高于对照组（p<0.0001 和 p<0.01，分别）。与对照组和 AMI 相比，SCAD 患者的 MPO 比活性更高（p<0.0001）。
该研究提出了一种用于在生物流体中测定 MPO 活性的稳健且敏感的方法，该方法具有低变异性。此外，比活性的测定可能为 MPO 在心血管疾病（CVDs）中的作用提供新的见解。
