She Z W, Mays D C, Sagone A L, Davis W B
Section of Pulmonary Diseases, Medical College of Georgia, Augusta 30912, USA.
Free Radic Biol Med. 1997;22(6):989-98. doi: 10.1016/s0891-5849(96)00486-8.
This study was designed to develop traps for hypochlorous acid (HOCl) which could be used to detect HOCl in the microenvironment of activated neutrophils. Reagent HOCl was found to react with para-aminobenzoic acid (PABA) in aqueous solution to produce a predominant metabolite detectable by high performance liquid chromatography (HPLC). Mass spectroscopy and nuclear magnetic resonance identified this metabolite as the ring addition product 3-chloro PABA. The related compound para-aminosalicylic acid (PAS) was also metabolized by HOCl to 3-chloro PAS. The formation of the 3-chloro metabolite was specific for reactions involving HOCl, since several other oxidants in chloride buffer failed to produce the metabolite. Human blood neutrophils activated by phorbol myristate acetate or zymosan in the presence of PABA (or PAS) used their HOCl to produce large amounts of the 3-chloro metabolite. The formation of 3-chloro PABA was inhibited by azide, catalase, and taurine, which is consistent with the production of the metabolite by the neutrophil myeloperoxidase (MPO) pathway. The reaction of HOCl with PABA and PAS was relatively slow as shown by competitive reactions with endogenous antioxidants like taurine, methionine, and glutathione. This was confirmed in reactions involving PABA/PAS and reagent HOCl or HOCl generated by the MPO enzyme system. In these in vitro systems, glutathione and serum completely inhibited the formation of the 3-chloro metabolite. In contrast, activated neutrophils metabolized PABA/PAS to the 3-chloro metabolite even in the presence of 1% serum. These findings demonstrate that PABA and PAS are specific trapping agents for HOCl produced by neutrophils in complex biological conditions.
本研究旨在开发用于次氯酸（HOCl）的捕获剂，该捕获剂可用于检测活化中性粒细胞微环境中的HOCl。研究发现，试剂HOCl在水溶液中与对氨基苯甲酸（PABA）反应，生成一种主要代谢产物，可通过高效液相色谱（HPLC）检测。质谱和核磁共振鉴定该代谢产物为环加成产物3-氯-PABA。相关化合物对氨基水杨酸（PAS）也被HOCl代谢为3-氯-PAS。3-氯代谢产物的形成是涉及HOCl反应所特有的，因为氯化物缓冲液中的其他几种氧化剂未能产生该代谢产物。在PABA（或PAS）存在下，被佛波酯肉豆蔻酸酯或酵母聚糖激活的人血中性粒细胞利用其HOCl产生大量的3-氯代谢产物。3-氯-PABA的形成受到叠氮化物、过氧化氢酶和牛磺酸的抑制，这与中性粒细胞髓过氧化物酶（MPO）途径产生该代谢产物一致。HOCl与PABA和PAS的反应相对较慢，如与牛磺酸、蛋氨酸和谷胱甘肽等内源性抗氧化剂的竞争反应所示。这在涉及PABA/PAS和试剂HOCl或MPO酶系统产生的HOCl的反应中得到了证实。在这些体外系统中，谷胱甘肽和血清完全抑制了3-氯代谢产物的形成。相比之下，即使在存在1%血清的情况下，活化的中性粒细胞也能将PABA/PAS代谢为3-氯代谢产物。这些发现表明，PABA和PAS是复杂生物条件下中性粒细胞产生的HOCl的特异性捕获剂。
