The Effect of Angeli's salt on Acute Hemolysis in a Canine Model

本研究的主要目的是确定全血各成分(血红蛋白、基质、血红蛋白+基质)在溶血过程中观察到的高血压中的作用，然后评价当归盐(Na2N2O32-)在犬急性血管内溶血模型中的治疗价值。一氧化氮(NO)是一种血管扩张剂，由血管内皮细胞持续产生。循环中可利用的NO的数量部分地受NO与血红蛋白结合的调节。血红蛋白通常包含在红细胞中，与一氧化氮的反应速度相对较慢。然而，循环内红细胞的破坏(血管内溶血)会导致血红蛋白(无细胞血红蛋白)从红细胞释放到循环中。在溶血过程中释放到循环中的无细胞血红蛋白与NO的结合速度比红细胞内的血红蛋白快得多。这种NO与无细胞血红蛋白的结合破坏了循环中可获得的NO的正常平衡，导致血管收缩，从而减少血流量，并导致器官损伤。虽然还不清楚基质(红细胞膜+红细胞膜的内容物)在溶血过程中起什么作用。这项研究将确定这一角色。此外，已知Angeli‘s盐能与血红蛋白迅速反应生成亚硝基血红蛋白Fe(II)NO，它不结合NO。 N_2O_3-(Angeli‘s盐)NO2-+NO NO+Fe(II)-O2(血浆含氧血红蛋白)Fe(III)(高铁血红蛋白)+ Fe(III)+NO-Fe(II)-NO(亚硝酸铁-血红蛋白) 如果Angeli盐可以阻止无细胞血红蛋白与NO结合，它可能会防止溶血过程中发生的血管收缩和由此导致的器官损伤。这项研究将使用我们成功开发并在前两种方案中使用的犬血管内溶血模型来测试Angeli盐在溶血过程中阻止NO与无细胞血红蛋白结合的能力。我们的模型使用自由水注入来创建血管内溶血，这模拟了人类急性血管内溶血的生理和生化特征。这种模式破坏了循环中的红细胞膜，导致血红蛋白释放到动物的循环中。在我们的模型中，急性血管内溶血导致血流动力学和器官功能的改变，即平均动脉压和全身血管阻力增加，心肾功能下降，分别以心脏指数和内生肌酐清除量衡量。 我们实验室以前的研究表明，升高的无细胞血红蛋白水平会消耗NO，吸入一氧化氮或静脉注射亚硝酸盐可以限制血管内溶血的有害影响。尽管这两种疗法都有明显的好处，但它们都有局限性。吸入一氧化氮价格昂贵，需要专门的输送系统，而且不易获得。静脉注射亚硝酸盐虽然便宜且易于管理，但与游离血红蛋白反应缓慢。这两种疗法都会产生高铁血红蛋白：(1)促进炎症，(2)与动脉粥样硬化有关，(3)可能发生还原并被回收为游离血红蛋白。另一方面，安吉利的盐已被证明能迅速与血红蛋白反应生成亚硝酰血红蛋白。与高铁血红蛋白(亚硝酸盐或NO与血红蛋白反应的产物)不同，亚硝酰血红蛋白相对稳定，没有已知的毒性。安吉丽的盐还有一个优势，那就是价格便宜，可以静脉注射。因此，Angeli‘s盐的化学特征表明，它应该与游离细胞血红蛋白发生不可逆转的反应，从而防止NO的消耗，可能会逆转血管内溶血的不利影响，并且比一氧化氮或亚硝酸盐治疗的副作用更少。 这项研究已经完成，目前正在对两份手稿进行审查后进行修订。 王丁，Pinowva B，所罗门SB，Cortes-PuchI，Kern SJ，Sun J，Gladwin MT，Helms C，Kim-Shapiro DB，Scheck hter an，Natanson C。在体内，无细胞高铁血红蛋白还原为氧合血红蛋白会导致犬血管收缩。提交的AJP，修订后 所罗门·SB，贝拉维亚·L，斯威尼·D，皮克诺娃B，佩拉加斯A，国王SB，拉特·NJH，克恩·SJ，孙杰，谢克特·安，娜塔森·C，格拉德温·MT，金-夏皮罗·DB。当归盐可对抗升高的血浆血红蛋白的血管活性作用。提交的FRBM，修订

The primary goal of this study is to determine the role of each component of whole blood (hemoglobin, stroma, hemoglobin + stroma) in the hypertension observed during hemolysis and then evaluate the therapeutic value of Angeli's salt (Na2N2O32-) in a canine model of acute intravascular hemolysis. Nitric oxide (NO) is a vasodilator which is constantly produced by the vascular endothelium. The amount of NO available in the circulation is, in part, regulated by the binding of NO to hemoglobin. Hemoglobin is normally contained within the red blood cell and reacts with nitric oxide at a relatively slow rate. However, the destruction of red blood cells within the circulation (intravascular hemolysis) causes the release of hemoglobin (cell-free hemoglobinh) from the red blood cell into the circulation. The cell-free hemoglobin released into the circulation during hemolysis binds to NO at a much faster rate than hemoglobin within the red blood cell. This binding of NO by cell-free hemoglobin disrupts the normal balance of NO available within the circulation resulting in vasoconstriction that decreases blood flow and leads to organ injury. Though it is not clear what role stroma (the contents of the red cell + the red cell membrane) has during hemolysis. This study will determine this role. In addition, Angeli's salt is known to react rapidly with hemoglobin to form, nitrosyl hemoglobin Fe(II)NO which does not bind NO. N2O3- (Angeli's Salt) NO2- + NO NO + Fe(II)-O2 (oxyhemoglobin in plasma) Fe(III) (methemoglobin) + Fe(III) + NO- Fe(II)-NO (iron-nitrosyl-hemoglobin) If the Angeli's salt can prevent the cell-free hemoglobin from binding NO, it may prevent the vasoconstriction and resulting organ injury that occurs during hemolysis. This study will test the ability of Angeli's salt to prevent cell-free hemoglobin binding of NO during hemolysis using the canine model of intravascular hemolysis that we successfully developed and used in two previous protocols. Our model uses a free water infusion to create intravascular hemolysis which mimics the physiologic and biochemical characteristics of acute intravascular hemolysis in the human. This model disrupts the red cell membrane within the circulation leading to the release of hemoglobin into circulation of the animal. In our model, acute intravascular hemolysis leads to changes in hemodynamics and organ function, i.e., increases in mean arterial pressure and systemic vascular resistance and decreases in heart and kidney function as measured by cardiac index and creatinine clearance respectively. Previous studies in our laboratory showed that elevated levels of cell-free hemoglobin consume NO, and therapy with inhaled nitric oxide or intravenous nitrite can limit the deleterious effects of intravascular hemolysis. Despite the demonstrated benefits, these two therapies have limitations. Inhaled nitric oxide is expensive, requires a specialized delivery system and is not readily available. Intravenous nitrite, although inexpensive and easy to administer, reacts slowly with cell free hemoglobin. Both therapies produce methemoglobin which: (1) promotes inflammation, (2) is associated with atherosclerosis and (3) can potentially undergo reduction and be recycled to free hemoglobin. Angeli's salt, on the other hand, has been shown to rapidly react with hemoglobin to produce nitrosyl hemoglobin. Unlike methemoglobin, (the product of nitrite or NO reacting with hemoglobin), nitrosyl hemoglobin is relatively stable and with no known toxicities. Angeli's salt also has the advantage of being inexpensive and can be administered intravenously. Thus, the chemical profile of Angeli's salt suggests that it should irreversibly react with cell free hemoglobin thereby preventing the consumption of NO, possibly reverse the adverse effects of intravascular hemolysis and have fewer side effects than either nitric oxide or nitrite therapy. This study has been completed and two manuscripts are currently being revised following review. Wang D, Piknova B, Solomon SB, Cortes-Puch I, Kern SJ, Sun J, Gladwin MT, Helms C, Kim-Shapiro DB, Schechter AN, Natanson C. In vivo reduction of cell-free methemoglobin to oxyhemoglobin results in vasoconstriction in canines. Submitted AJP, Revised Solomon SB, Bellavia L, Sweeney D, Piknova B, Perlagas A, King SB, Raat NJH, Kern SJ, Sun J, Schechter AN, Natanson C, Gladwin MT, Kim-Shapiro DB. Angelis Salt Counteracts the Vasoactive Effects of Elevated Plasma Hemoglobin. Submitted FRBM, Revised