Computation of plasma hemoglobin nitric oxide scavenging in hemolytic anemias

Abstract

Intravascular hemoglobin limits the amount of endothelial-derived nitric oxide (NO) available for vasodilation. Cell-free hemoglobin scavenges NO more efficiently than red blood cell-encapsulated hemoglobin. Hemolysis has recently been suggested to contribute to endothelial dysfunction based on a mechanism of NO scavenging by cell-free hemoglobin. Although experimental evidence for this phenomenon has been presented, support from a theoretical approach has, until now, been missing. Indeed, due to the low amounts of cell-free hemoglobin present in these pathological conditions, the role of cell-free hemoglobin scavenging of NO in disease has been questioned. In this study, we model the effects of cell-free hemoglobin on NO bioavailability, focusing on conditions that closely mimic those under known pathological conditions. We find that as little as 1 microM cell-free intraluminal hemoglobin (heme concentration) can significantly reduce NO bioavailability. In addition, extravasation of hemoglobin out of the lumen has an even greater effect. We also find that low hematocrit associated with anemia increases NO bioavailability but also leads to increased susceptibility to NO scavenging by cell-free hemoglobin. These results support the paradigm that cell-free hemoglobin released into plasma during intravascular hemolysis in human disease contributes to the experimentally observed reduction in NO bioavailability and endothelial dysfunction.

Copyright 2006 Elsevier Inc.

Figures

Figure 1

Illustration of regions modeled. RBCs are in the lumen of the blood vessel, which includes a red blood cell-free zone. NO is produced in the endothelium which is on the exterior of the blood vessel. The smooth muscle is the outermost layer around the blood vessel. The rate of NO production, scavenging, and diffusion is defined separately for each compartment as described in the materials and methods section. For example, Equation 4, only applies inside of the individual RBCs. This schematic of the modeled regions is not drawn to scale.

Figure 2

NO bioavailability dependence on hematocrit and cell-free zone. Calculated NO concentrations versus the distance from the center of the blood vessel are plotted for Hct values of 18%, 25%, and 45% with and without a cell-free zone. The position of the endothelium is shown by the bent arrow at 0.05 mm. A 2.5 μm thick region in the vessel closest to the endothelium was free of red blood cells in the cases having a cell-free zone indicated by solid lines. RBCs were placed within the 2.5 μm region for the scenarios without a cell-free zone indicated by solid lines with markers. Total Hb in the lumen due to RBCs was set to 4.14 mM for 18% Hct, 5.75 mM for 25% Hct, and 10.35 mM for 45%. NO was produced at a rate of 42.4 μM s−1 in the endothelium. The RBC Pm was set to 450 μms−1.

Figure 3

Effect of RBC membrane permeability and cell-free Hb on [NO]. Concentrations of NO for high and low cell membrane permeability at 44% Hct are plotted versus the distance along the vessel axis. The permeability of the cell membrane was set to 9 × 105 μm s−1 for high permeability (solid lines with no markers) and to 450 μm s−1 for low permeability (solid lines with markers). Concentrations of cell-free Hb ([Hbcf]) were 0, 1, 4, 10, 20, and100 μM (a single color was used for each concentration of cell-free Hb for both high and low permeability). Total RBC encapsulated Hb in the lumen was 10 mM. A 2.5 μm cell free zone was included in these and all subsequent computations, however cell-free Hb was allowed to enter this region. The effect of the RBC membrane permeability on NO levels is imperceptible at a cell-free Hb concentration of 4 μM and virtually eliminated for cases with concentrations of 5 μM cell-free Hb and above.

Figure 4

Concentration of NO as a function of extravasated cell-free Hb for conditions present in sickle cell disease. The concentrations of NO at particular distances from the center of the vessel are plotted as a function of extravasated Hb. Hematocrits of 18% and 25% correspond to values found during crisis and steady state in SCD, respectively. A Hct of 50% was used for comparison to normal physiology. Cell-free Hb concentrations ([Hbcf]) were set to 4 and 25 μM. a) NO concentrations at 55.6 μm from the center of the blood vessel. Results for 18% Hct (pink square), 25% Hct (yellow triangle), and 50% (purple plus sign) with 4 μM cell-free Hb were the same so that curves representing these overlap each other. Results for 18% Hct (blue diamond), 25% Hct (teal x), and 50% (red circle) with 25 μM cell-free Hb also lead to the same levels of NO as each other and thus overlap. b) the concentration of NO at 55.6 μm in the absence of extravasation with 4 and 25 μM cell-free Hb is plotted at different Hcts (18%, 25%, and 50%). c) NO concentrations at 500 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. d) NO concentrations at 975 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated for the above conditions. Insets: illustration of the position where [NO] was measured (refer to Figure 1 for labeled regions). Insets are not drawn to scale.

Figure 4

Concentration of NO as a function of extravasated cell-free Hb for conditions present in sickle cell disease. The concentrations of NO at particular distances from the center of the vessel are plotted as a function of extravasated Hb. Hematocrits of 18% and 25% correspond to values found during crisis and steady state in SCD, respectively. A Hct of 50% was used for comparison to normal physiology. Cell-free Hb concentrations ([Hbcf]) were set to 4 and 25 μM. a) NO concentrations at 55.6 μm from the center of the blood vessel. Results for 18% Hct (pink square), 25% Hct (yellow triangle), and 50% (purple plus sign) with 4 μM cell-free Hb were the same so that curves representing these overlap each other. Results for 18% Hct (blue diamond), 25% Hct (teal x), and 50% (red circle) with 25 μM cell-free Hb also lead to the same levels of NO as each other and thus overlap. b) the concentration of NO at 55.6 μm in the absence of extravasation with 4 and 25 μM cell-free Hb is plotted at different Hcts (18%, 25%, and 50%). c) NO concentrations at 500 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. d) NO concentrations at 975 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated for the above conditions. Insets: illustration of the position where [NO] was measured (refer to Figure 1 for labeled regions). Insets are not drawn to scale.

Figure 4

Concentration of NO as a function of extravasated cell-free Hb for conditions present in sickle cell disease. The concentrations of NO at particular distances from the center of the vessel are plotted as a function of extravasated Hb. Hematocrits of 18% and 25% correspond to values found during crisis and steady state in SCD, respectively. A Hct of 50% was used for comparison to normal physiology. Cell-free Hb concentrations ([Hbcf]) were set to 4 and 25 μM. a) NO concentrations at 55.6 μm from the center of the blood vessel. Results for 18% Hct (pink square), 25% Hct (yellow triangle), and 50% (purple plus sign) with 4 μM cell-free Hb were the same so that curves representing these overlap each other. Results for 18% Hct (blue diamond), 25% Hct (teal x), and 50% (red circle) with 25 μM cell-free Hb also lead to the same levels of NO as each other and thus overlap. b) the concentration of NO at 55.6 μm in the absence of extravasation with 4 and 25 μM cell-free Hb is plotted at different Hcts (18%, 25%, and 50%). c) NO concentrations at 500 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. d) NO concentrations at 975 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated for the above conditions. Insets: illustration of the position where [NO] was measured (refer to Figure 1 for labeled regions). Insets are not drawn to scale.

Figure 4

Concentration of NO as a function of extravasated cell-free Hb for conditions present in sickle cell disease. The concentrations of NO at particular distances from the center of the vessel are plotted as a function of extravasated Hb. Hematocrits of 18% and 25% correspond to values found during crisis and steady state in SCD, respectively. A Hct of 50% was used for comparison to normal physiology. Cell-free Hb concentrations ([Hbcf]) were set to 4 and 25 μM. a) NO concentrations at 55.6 μm from the center of the blood vessel. Results for 18% Hct (pink square), 25% Hct (yellow triangle), and 50% (purple plus sign) with 4 μM cell-free Hb were the same so that curves representing these overlap each other. Results for 18% Hct (blue diamond), 25% Hct (teal x), and 50% (red circle) with 25 μM cell-free Hb also lead to the same levels of NO as each other and thus overlap. b) the concentration of NO at 55.6 μm in the absence of extravasation with 4 and 25 μM cell-free Hb is plotted at different Hcts (18%, 25%, and 50%). c) NO concentrations at 500 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. d) NO concentrations at 975 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated for the above conditions. Insets: illustration of the position where [NO] was measured (refer to Figure 1 for labeled regions). Insets are not drawn to scale.

Figure 5

Concentration of NO as a function of extravasated cell-free Hb for conditions present in paroxysmal nocturnal hemoglobinuria. The concentrations of NO at particular distances from the center of the vessel are plotted as a function of extravasated Hb. Cell-free Hb concentrations ([Hbcf]) were set to 30, 125, and 625 μM and concentrations of cell-free Hb extravasated into the endothelium ranged from 0–10 μM for 25% Hct. A Hct of 50%, found under normal conditions, was used for comparison. a) NO concentrations at 55.6 μm from the center of the blood vessel are shown. Curves using 25% Hct (blue diamond) and 50% Hct (teal x) with 30 μM cell-free Hb overlap each other. Curves using 25% Hct (pink square) and 50% Hct (purple plus sign) with 125 μM cell-free Hb also lead to the same levels of NO. Similarly, 25% Hct (yellow triangle) and 50% Hct (red circle) with 625 μM cell-free Hb give rise to the same concentrations of NO. b) NO concentrations at 500 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. c) NO concentrations at 975 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. Insets: illustration of the position where [NO] was measured (refer to Figure 1 for labeled regions). Insets are not drawn to scale.

Figure 5

Concentration of NO as a function of extravasated cell-free Hb for conditions present in paroxysmal nocturnal hemoglobinuria. The concentrations of NO at particular distances from the center of the vessel are plotted as a function of extravasated Hb. Cell-free Hb concentrations ([Hbcf]) were set to 30, 125, and 625 μM and concentrations of cell-free Hb extravasated into the endothelium ranged from 0–10 μM for 25% Hct. A Hct of 50%, found under normal conditions, was used for comparison. a) NO concentrations at 55.6 μm from the center of the blood vessel are shown. Curves using 25% Hct (blue diamond) and 50% Hct (teal x) with 30 μM cell-free Hb overlap each other. Curves using 25% Hct (pink square) and 50% Hct (purple plus sign) with 125 μM cell-free Hb also lead to the same levels of NO. Similarly, 25% Hct (yellow triangle) and 50% Hct (red circle) with 625 μM cell-free Hb give rise to the same concentrations of NO. b) NO concentrations at 500 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. c) NO concentrations at 975 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. Insets: illustration of the position where [NO] was measured (refer to Figure 1 for labeled regions). Insets are not drawn to scale.

Figure 5

Concentration of NO as a function of extravasated cell-free Hb for conditions present in paroxysmal nocturnal hemoglobinuria. The concentrations of NO at particular distances from the center of the vessel are plotted as a function of extravasated Hb. Cell-free Hb concentrations ([Hbcf]) were set to 30, 125, and 625 μM and concentrations of cell-free Hb extravasated into the endothelium ranged from 0–10 μM for 25% Hct. A Hct of 50%, found under normal conditions, was used for comparison. a) NO concentrations at 55.6 μm from the center of the blood vessel are shown. Curves using 25% Hct (blue diamond) and 50% Hct (teal x) with 30 μM cell-free Hb overlap each other. Curves using 25% Hct (pink square) and 50% Hct (purple plus sign) with 125 μM cell-free Hb also lead to the same levels of NO. Similarly, 25% Hct (yellow triangle) and 50% Hct (red circle) with 625 μM cell-free Hb give rise to the same concentrations of NO. b) NO concentrations at 500 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. c) NO concentrations at 975 μm from the center of the blood vessel versus concentration of cell-free Hb extravasated. Insets: illustration of the position where [NO] was measured (refer to Figure 1 for labeled regions). Insets are not drawn to scale.