Heart Research Institute, The University of Sydney, Sydney, New South Wales, Australia.
School of Chemistry, The University of Sydney, Sydney, New South Wales, Australia; Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, New South Wales, Australia.
Free Radic Biol Med. 2023 Feb 1;195:23-35. doi: 10.1016/j.freeradbiomed.2022.12.014. Epub 2022 Dec 22.
Conversion of the redox probe hydroethidine (HE) to 2-chloroethidium (2-Cl-E) by myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) provides comparable specificity and superior sensitivity to measurement of 3-chlorotyrosine (3-Cl-Tyr), the gold standard biomarker for MPO chlorinating activity in biological systems. However, a limitation of the former method is the complex mixture of products formed by the reaction of HE with reagent HOCl, coupled with the difficult purification of 2-Cl-E from this mixture for analytical purposes. This limitation prompted us to test whether 2-Cl-E could be formed by reaction of HE with the strong and widely used chlorinating agent, N-chlorosuccinimide (NCS). Unexpectedly, such reaction yielded 2-chlorohydroethidine (2-Cl-HE) as the major product in addition to 2-Cl-E, as assessed by high performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR). 2-Cl-HE was also observed to be the major chlorination product formed from HE with both reagent and enzymatically generated HOCl, just as it was formed ex vivo in different healthy and diseased mouse and human tissues upon incubation with glucose/glucose oxidase to generate a flux of hydrogen peroxide (HO). Quantification of 2-Cl-HE plus 2-Cl-E improved the sensitivity of the HE-based method compared with measurement of only 2-Cl-E. Moreover, 2-chlorodimidium (2-Cl-D) was developed as a practical internal standard instead of the previously used internal standard, deuterated 2-Cl-E (d-2-Cl-E). Overall, the present study describes an improved method for the detection of MPO/chlorinating activity in biological systems of health and disease.
过氧化物酶（MPO）衍生的次氯酸（HOCl）将氧化还原探针羟乙基（HE）转化为 2-氯乙基（2-Cl-E），可提供与 3-氯酪氨酸（3-Cl-Tyr）测量相当的特异性和更高的灵敏度，3-Cl-Tyr 是生物系统中 MPO 氯化活性的金标准生物标志物。然而，前一种方法的局限性在于 HE 与试剂 HOCl 反应形成的产物混合物复杂，并且由于难以从该混合物中纯化 2-Cl-E 用于分析目的，因此存在局限性。这一限制促使我们测试 HE 是否可以与强而广泛使用的氯化剂 N-氯代丁二酰亚胺（NCS）反应形成 2-Cl-E。出乎意料的是，正如高效液相色谱（HPLC）、质谱（MS）和核磁共振（NMR）所评估的那样，这种反应除了 2-Cl-E 之外，还主要生成 2-氯羟乙基（2-Cl-HE）。还观察到 2-Cl-HE 是 HE 与试剂和酶促生成的 HOCl 形成的主要氯化产物，就像在不同的健康和患病的小鼠和人组织中孵育时与葡萄糖/葡萄糖氧化酶一起形成的那样，以产生过氧化氢（HO）的通量。与仅测量 2-Cl-E 相比，量化 2-Cl-HE 加 2-Cl-E 提高了基于 HE 的方法的灵敏度。此外，开发了 2-氯二脒（2-Cl-D）作为实用的内标，而不是以前使用的内标氘代 2-Cl-E（d-2-Cl-E）。总的来说，本研究描述了一种用于检测健康和疾病生物系统中 MPO/氯化活性的改进方法。
