From the Vascular Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia.
J Biol Chem. 2014 Feb 28;289(9):5580-95. doi: 10.1074/jbc.M113.539486. Epub 2014 Jan 16.
Oxidants derived from myeloperoxidase (MPO) contribute to inflammatory diseases. In vivo MPO activity is commonly assessed by the accumulation of 3-chlorotyrosine (3-Cl-Tyr), although 3-Cl-Tyr is formed at low yield and is subject to metabolism. Here we show that MPO activity can be assessed using hydroethidine (HE), a probe commonly employed for the detection of superoxide. Using LC/MS/MS, (1)H NMR, and two-dimensional NOESY, we identified 2-chloroethidium (2-Cl-E(+)) as a specific product when HE was exposed to hypochlorous acid (HOCl), chloramines, MPO/H2O2/chloride, and activated human neutrophils. The rate constant for HOCl-mediated conversion of HE to 2-Cl-E(+) was estimated to be 1.5 × 10(5) M(-1)s(-1). To investigate the utility of 2-Cl-E(+) to assess MPO activity in vivo, HE was injected into wild-type and MPO-deficient (Mpo(-/-)) mice with established peritonitis or localized arterial inflammation, and tissue levels of 2-Cl-E(+) and 3-Cl-Tyr were then determined by LC/MS/MS. In wild-type mice, 2-Cl-E(+) and 3-Cl-Tyr were detected readily in the peritonitis model, whereas in the arterial inflammation model 2-Cl-E(+) was present at comparatively lower concentrations (17 versus 0.3 pmol/mg of protein), and 3-Cl-Tyr could not be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E(+) were decreased substantially in Mpo(-/-) mice, indicative of the specificity of the assay. In the arterial inflammation model, 2-Cl-E(+) was absent from non-inflamed arteries and blood, suggesting that HE oxidation occurred locally in the inflamed artery. Our data suggest that the conversion of exogenous HE to 2-Cl-E(+) may be a useful selective and sensitive marker for MPO activity in addition to 3-Cl-Tyr.
髓过氧化物酶（MPO）衍生的氧化剂有助于炎症性疾病。体内 MPO 活性通常通过 3-氯酪氨酸（3-Cl-Tyr）的积累来评估，尽管 3-Cl-Tyr 的形成产率低且易受代谢影响。在这里，我们展示了可以使用羟乙基（HE）评估 MPO 活性，HE 是一种常用于检测超氧化物的探针。通过 LC/MS/MS、（1）H NMR 和二维 NOESY，我们确定当 HE 暴露于次氯酸（HOCl）、氯胺、MPO/H2O2/氯化物和激活的人中性粒细胞时，会产生 2-氯乙啶（2-Cl-E(+)）作为特定产物。HOCl 介导的 HE 转化为 2-Cl-E(+)的速率常数估计为 1.5×10(5) M(-1)s(-1)。为了研究 2-Cl-E(+)在体内评估 MPO 活性的实用性，将 HE 注射到患有已建立的腹膜炎或局部动脉炎症的野生型和 MPO 缺陷型（Mpo(-/-)）小鼠中，然后通过 LC/MS/MS 测定组织中 2-Cl-E(+)和 3-Cl-Tyr 的水平。在野生型小鼠中，在腹膜炎模型中很容易检测到 2-Cl-E(+)和 3-Cl-Tyr，而在动脉炎症模型中，2-Cl-E(+)的浓度相对较低（17 与 0.3 pmol/mg 蛋白质），并且无法检测到 3-Cl-Tyr。与 3-Cl-Tyr 的情况类似，在 Mpo(-/-)小鼠中，组织中 2-Cl-E(+)的水平大大降低，表明该测定具有特异性。在动脉炎症模型中，未发炎的动脉和血液中不存在 2-Cl-E(+)，表明 HE 的氧化是在发炎的动脉局部发生的。我们的数据表明，外源性 HE 转化为 2-Cl-E(+)可能是 MPO 活性的一种有用的选择性和敏感性标志物，除了 3-Cl-Tyr 之外。
