Daniel B. Kim-Shapiro

Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation.

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extra-erythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases.

Concerted nitric oxide formation and release from the simultaneous reactions of nitrite with deoxy- and oxyhemoglobin

Recent studies reveal a novel role for hemoglobin as an allosterically regulated nitrite reductase that may mediate nitric oxide (NO)-dependent signaling along the physiological oxygen gradient. Nitrite reacts with deoxyhemoglobin in an allosteric reaction that generates NO and oxidizes deoxyhemoglobin to methemoglobin. NO then reacts at a nearly diffusion-limited rate with deoxyhemoglobin to form iron-nitrosyl-hemoglobin, which to date has been considered a highly stable adduct and, thus, not a source of bioavailable NO. However, under physiological conditions of partial oxygen saturation, nitrite will also react with oxyhemoglobin, and although this complex autocatalytic reaction has been studied for a century, the interaction of the oxy- and deoxy-reactions and the effects on NO disposition have never been explored. We have now characterized the kinetics of hemoglobin oxidation and NO generation at a range of oxygen partial pressures and found that the deoxy-reaction runs in parallel with and partially inhibits the oxy-reaction. In fact, intermediates in the oxy-reaction oxidize the heme iron of ironnitrosyl-hemoglobin, a product of the deoxy-reaction, which releases NO from the iron-nitrosyl. This oxidative denitrosylation is particularly striking during cycles of hemoglobin deoxygenation and oxygenation in the presence of nitrite. These chemistries may contribute to the oxygen-dependent disposition of nitrite in red cells by limiting oxidative inactivation of nitrite by oxyhemoglobin, promoting nitrite reduction to NO by deoxyhemoglobin, and releasing free NO from iron-nitrosyl-hemoglobin.

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

Release of hemoglobin from the erythrocyte during intravascular hemolysis contributes to the pathology of a variety of diseased states. This effect is partially due to the enhanced ability of cell-free plasma hemoglobin, which is primarily found in the ferrous, oxygenated state, to scavenge nitric oxide. Oxidation of the cell-free hemoglobin to methemoglobin, which does not effectively scavenge nitric oxide, using inhaled nitric oxide has been shown to be effective in limiting pulmonary and systemic vasoconstriction. However, the ferric heme species may be reduced back to ferrous hemoglobin in plasma and has the potential to drive injurious redox chemistry. We propose that compounds that selectively convert cell-free hemoglobin to ferric, and ideally iron-nitrosylated heme species that do not actively scavenge nitric oxide, would effectively treat intravascular hemolysis. We show here that nitroxyl generated by Angelis salt (sodium alpha-oxyhyponitrite, Na2N2O3) preferentially reacts with cell-free hemoglobin compared to that encapsulated in the red blood cell under physiologically relevant conditions. Nitroxyl oxidizes oxygenated ferrous hemoglobin to methemoglobin and can convert the methemoglobin to a more stable, less toxic species, iron-nitrosyl hemoglobin. These results support the notion that Angelis salt or a similar compound could be used to effectively treat conditions associated with intravascular hemolysis.