NO or No NO, Increased Reduction of Nitrite to Nitric Oxide by Modified Red Blood Cells

Nitric oxide (NO) metabolism is dysregulated in sickle cell disease (SCD) and Thalassemia. Both higher consumption of arginine and arginase-released by hemolysis are implicated in decreased production of NO, negatively affecting vaso-dilation and resolve of vaso-occlusive crises (VOC). The Red Blood Cell (RBC) plays a role in NO transport, as well as in nitrite (NO₂⁻) reduction to NO. We studied the relation between oxygen affinity of RBCs and their capacity to reduce NO₂⁻ to NO under different partial oxygen pressures.

Blood samples were collected with IRB approval from normal controls, b-thalassamia (bThal) patients, and SCD patients including those who were treated with hydroxyurea (HU). RBC hemoglobin (Hb) was modified with compounds that increased the oxygen affinity (lowering P₅₀) including anti-sickling agents such as 5-hydroxymethyl-2-furfural (5HMF). Samples at different hematocrits, with different fractions of modified RBCs or free Hb, were incubated in a Tonometer (Instrumentation Laboratory) at 37°C under a constant flow of an Air/Nitrogen gas mixture. Varying amounts of NO₂⁻ were added to the cells, and NO₂⁻ and NO₃⁻ were measured in the extracellular medium. The gas outflow from the Tonometer was collected in mylar balloons, and NO production was measured using a Nitric Oxide Analyzer (GE Analytical Instruments). Before and after incubation the samples were analyzed by spectroscopy, and the oxygen affinity was measured with the Hemox Analyzer (TCS Medical Products).

The concentration of NO₂⁻ decreased exponentially in the extracellular fluid with a rate that increased with hematocrit, and oxygen tension to reach an apparent equilibrium after 30–45 minutes. NO₃⁻, but little NO, from any of the RBC mixtures was released under room air. NO was generated under nitrogen from all RBC mixtures, and the rate and amounts released in 30 minutes were directly related to NO₂⁻ concentration, hematocrit and fraction of free Hb present. High levels of NO₂⁻ resulted in irreversible modification of RBCs and a loss of oxygen carrying capacity as measured by spectroscopy and oxygen affinity analysis. When these cells were mixed with unmodified RBCs, NO production was related to the fraction of RBCs with an altered P₅₀. Hb modification in normal and bThal cells increased NO production with decreasing P₅₀. SCD-RBCs showed a higher NO generation as compared to normal RBCs, which increased with the presence of HbF. Modification of SCD-RBCs further increased NO production with a decreased P₅₀. Together, any decrease (left-shift) in P₅₀ resulted in an increase in NO production under low oxygen conditions. NO formation was dependent on the subpopulation of modified cells in the entire population.

Any condition that decreased the P₅₀, resulted in an increased reduction of NO₂⁻ to NO under low oxygen tension. These modifications include potential, and reported anti-sickling agents such as HU and 5HMF. In addition, mixing extensively modified RBCs or free Hb with untreated RBCs increased the capacity of the entire population to reduce NO₂⁻. Interestingly, SCD-RBCs with low levels of HbF, which showed a slightly increased P₅₀ as compared to normal RBCs, showed an increased ability to reduce NO₂⁻. We hypothesize that the local presence of modified RBCs with an increased oxygen affinity or the presence of free Hb will increase the local formation of NO from NO₂⁻. As such, anti-sickling agents may be beneficial to lower the incidence of VOC, not only by reducing the rate of Hb polymerization, but also by the increased capacity of the RBCs to generate NO for vaso-dilation.

Fitch:RadioRx, Inc: Research Funding. Scicinski:RadioRx, Inc: Research Funding. Oronsky:RadioRx, Inc: Research Funding.