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

安吉利盐对犬模型急性溶血的影响

• 批准号: 8952826
• 负责人: Charles Natanson
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8952826
• 关键词: Acute; Animals; Atherosclerosis; Binding; Biochemical; Blood; Blood Circulation; Blood Pressure; Blood flow; Breathing; Canis familiaris; Cardiac; Cell membrane; Cells; Cessation of life; Characteristics; Creatinine clearance measurement; Data; Endothelium; Equilibrium; Erythrocytes; Gases; Gastrointestinal tract structure; Goals; Health; Heart; Hemoglobin; Hemolysis; Human; Hypertension; Inflammation; Infusion procedures; Injury; Intravenous; Kidney; Laboratories; Measures; Methemoglobin; Modeling; Nitric Oxide; Nitric Oxide Donors; Nitrites; Organ; Oxidation-Reduction; Oxyhemoglobin; Paper; Peripheral; Peripheral Resistance; Physiological; Plasma; Property; Protocols documentation; Publishing; Pulmonary Vascular Resistance; Reaction; Recycling; Renal function; Risk; Role; Series; Smooth Muscle; Sodium Chloride; System; Testing; Therapeutic; Tissues; Vascular Endothelium; Vasodilation; Vasodilator Agents; Water; Whole Blood; design; heart function; hemodynamics; indexing; inhaled nitric oxide; iron nitrosyl; nitrosyl hemoglobin; pressure; prevent; vasoconstriction

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 thought not thought to be vasoactive promotes inflammation, is associated with atherosclerosis and can potentially undergo reduction and be recycled to free hemoglobin. When analyzing the effects of Angeli's salt, the action seems to have been mainly, or at least largely, due to its ability to vasodilate. Indeed, infusion of Angeli's Salt in the absence of hemolysis in this study led to substantial effects associated vasodilation whereas NO administration in the absence of hemolysis did not have this effect. In addition, while Angeli's Salt only resulted in conversion of about 20% of plasma Hb to metHb, NO inhalation led to about 80% conversion.40 Thus, both approaches could alleviate hemolysis-dependent vasoconstriction through both inactivation of vasoconstricting oxyHb and by compensatory vasodilating effects, but the degree to each of these contributes is different for each treatment. To better understand the role of cell free oxyHb and metHb in these reactions, we designed a study to determine the effects of cell-free metHb on vasocontriction and redox reactions. Our study suggest that both forms of cell-free hemoglobin are able to extravasate through endothelium and show vasoactivity outside the luminal space. We suspect that NO destruction outside of the luminal space, in the smooth muscle tissue, has a bigger effect on the observed mean blood pressure than changes in the lumen or that hemoglobin and its metabolites in tissues also have significant effects. Our study showed that cell-free metHb in the bloodstream should be considered as vasoactive substance, as it is converted into oxyHb, which can increase blood pressure by scavenging NO in the blood or tissues or by some still unclear mechanism. This series of studies has resulted in 4 published papers

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，而高铁血红蛋白被认为不具有血管活性，会促进炎症，与动脉粥样硬化有关，可能会被减少，并被回收利用，以产生游离的血红蛋白。当分析Angeli盐的效果时，这种作用似乎主要或至少在很大程度上是由于它的血管扩张能力。事实上，在本研究中，在没有溶血的情况下输注Angeli‘s盐导致了与血管扩张相关的实质性效果，而在没有溶血的情况下没有给药则没有这种效果。此外，虽然Angeli‘s盐只导致大约20%的血浆Hb转化为metHb，但吸入NO导致大约80%的转化。40因此，这两种方法都可以通过灭活血管收缩氧合Hb和代偿血管扩张作用来缓解溶血依赖的血管收缩，但每种治疗的贡献程度都不同。 为了更好地了解游离氧合Hb和metHb在这些反应中的作用，我们设计了一项研究，以确定无细胞metHb对血管收缩和氧化还原反应的影响。我们的研究表明，这两种形式的无细胞血红蛋白都能通过血管内皮细胞外渗，并显示管腔外的血管活性。我们怀疑，在管腔以外的组织中，没有任何破坏比管腔的变化对观察到的平均血压有更大的影响，或者组织中的血红蛋白及其代谢物也有显著的影响。我们的研究表明，血流中的无细胞metHb应该被认为是血管活性物质，因为它被转化为oxHb，它可以通过清除血液或组织中的NO或某些尚不清楚的机制来升高血压。 这一系列研究已发表了4篇论文。