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

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Abstract

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