Hemolysis-associated endothelial dysfunction mediated by accelerated NO inactivation by decompartmentalized oxyhemoglobin

Abstract

During intravascular hemolysis in human disease, vasomotor tone and organ perfusion may be impaired by the increased reactivity of cell-free plasma hemoglobin (Hb) with NO. We experimentally produced acute intravascular hemolysis in a canine model in order to test the hypothesis that low levels of decompartmentalized or cell-free plasma Hb will severely reduce NO bioavailability and produce vasomotor instability. Importantly, in this model the total intravascular Hb level is unchanged; only the compartmentalization of Hb within the erythrocyte membrane is disrupted. Using a full-factorial design, we demonstrate that free water-induced intravascular hemolysis produces dose-dependent systemic vasoconstriction and impairs renal function. We find that these physiologic changes are secondary to the stoichiometric oxidation of endogenous NO by cell-free plasma oxyhemoglobin. In this model, 80 ppm of inhaled NO gas oxidized 85-90% of plasma oxyhemoglobin to methemoglobin, thereby inhibiting endogenous NO scavenging by cell-free Hb. As a result, the vasoconstriction caused by acute hemolysis was attenuated and the responsiveness to systemically infused NO donors was restored. These observations confirm that the acute toxicity of intravascular hemolysis occurs secondarily to the accelerated dioxygenation reaction of plasma oxyhemoglobin with endothelium-derived NO to form bioinactive nitrate. These biochemical and physiological studies demonstrate a major role for the intact erythrocyte in NO homeostasis and provide mechanistic support for the existence of a human syndrome of hemolysis-associated NO dysregulation, which may contribute to the vasculopathy of hereditary, acquired, and iatrogenic hemolytic states.

Figure 1

Characteristics of the canine free water hemolysis model. (A) Increasing rates of free water infusion caused increased rates of hemolysis with higher total plasma Hb levels (concentration in terms of heme groups). (B) The amount of plasma Hb (concentration in terms of heme groups) released by free water–induced intravascular hemolysis remained ferrous (HbFe^II-O₂) and correlated with the ability of the plasma to consume NO in a 1:1 stoichiometric ratio (r = 0.98; P < 0.001). The inset represents a sample spectrum of the Hb species in the plasma, which demonstrates that the cell-free plasma Hb consists predominantly of oxyhemoglobin. (C and D) Compared with 6-hour control infusions (D5W and normal saline combined), 6-hour infusions of free water caused a significant increase in MAP (C; P = 0.0004) and a significant decrease in 6-hour creatinine clearance (D; P = 0.03) from time 0.

Figure 2

Full-factorial study design of the effects of intravascular hemolysis and inhaled NO. In order to minimize variability and limit the number of animals necessary to perform these studies, each animal underwent a baseline and intervention experiment. During the first week, each animal underwent a 6-hour baseline study with an infusion of D5W to control for the effects of the fluid challenge. During the second week, animals underwent a 6-hour intervention study in which they were randomized to receive 1 of 4 treatments (D5W; D5W plus inhaled NO; free water; or free water plus inhaled NO). This design allowed for the comparison of differences across treatment groups by subtracting calculated differences within animals (from baseline to intervention) in each treatment group. Comparison of these differences of the differences allowed for analysis of the effects of hemolysis, the effects of inhaled NO, and detection of any interaction between the 2 interventions.

Figure 3

Effects of hemolysis and inhaled NO on MAP. In paired experiments, all animals received a 6-hour D5W infusion during the baseline study and 1 week later were randomized to a 6-hour intervention study of either D5W, D5W plus NO, free water, or free water plus NO. Changes in MAP over the course of the 6-hour baseline (filled circles) and intervention studies (open circles) are shown. In all 4 groups of animals, there were statistically similar small increases in MAP during the 6-hour baseline D5W infusion. Compared with an equivalent infusion of D5W with or without NO (nonhemolyzing control groups), free water–induced intravascular hemolysis caused a significant increase in MAP, which was attenuated by the concurrent inhalation of NO gas (P = 0.0003 for interaction of NO and hemolysis).

Figure 4

Relationship between total cell-free plasma Hb and the physiologic effects of hemolysis and inhaled NO. Upper panels: The difference in response from 0 to 6 hours between baseline and intervention studies for each of the 4 treatment groups is shown for MAP and SVRI. In animals receiving D5W (nonhemolyzing control groups), inhaled NO had no net effect on MAP and SVRI. Compared with these nonhemolyzing controls, free water–induced intravascular hemolysis caused significant increases in MAP and SVRI, which were attenuated by the concurrent inhalation of NO gas (P = 0.0003 for interaction of NO and hemolysis for both variables). Lower panels: Relationship between change in MAP and SVRI and total plasma Hb levels (concentration in terms of heme groups) during the intervention studies in the hemolyzing groups (free water and free water plus NO groups). Despite similar total plasma Hb levels in these 2 groups, the relationships between change in MAP and SVRI and total plasma Hb levels were significantly different (P = 0.003 and P = 0.001, respectively). As total plasma Hb levels increased, MAP and SVRI increased more in the free water group than in the free water plus NO group.

Figure 5

Effects of hemolysis and inhaled NO on renal function. (A) The difference in response from 0 to 6 hours between baseline and intervention studies for each of the 4 treatment groups is shown for serum sodium levels. Compared with infusion of D5W with or without NO, free water–induced intravascular hemolysis caused a significant impairment in the ability of the kidneys to compensate for hyponatremia, which was attenuated by the concurrent inhalation of NO (P = 0.04). (B) Six-hour creatinine clearance values during the intervention studies for each of the 4 treatment groups are shown. Based on a priori hypotheses, the creatinine clearance values ordered as expected, with the free water group having the lowest clearance, the D5W and D5W plus NO groups having the highest clearances, and the free water plus NO group having an intermediate clearance approaching that of the D5W and D5W plus NO groups (P = 0.01).

Figure 6

Plasma NO consumption and plasma Hb levels. (A) A significantly different relationship exists between plasma NO consumption and total plasma Hb levels (concentration in terms of heme groups) in the free water and free water plus NO groups (P < 0.0001). The inset demonstrates the relationships over the entire range of measured Hb levels, whereas the main graph focuses on the physiologic range of hemolysis in human disease states. (B) Spectral deconvolution of the plasma Hb species. The upper spectrum represents reference tracings for canine oxyhemoglobin and methemoglobin. The middle and lower spectra represent characteristic samples from the free water and free water plus NO treatment groups, respectively. (C) Total plasma Hb composition in the free water and free water plus NO groups was significantly different at 6 hours (P = 0.03). In the free water group, the plasma contained predominantly oxyhemoglobin. In contrast, in the free water plus NO group, the plasma contained predominantly methemoglobin.

Figure 7

Physiologic effects of sodium nitroprusside during hemolysis with and without inhaled NO. Percent change in SVRI (A) and CI (B) in response to increasing doses of sodium nitroprusside during the intervention studies for each of the 4 treatment groups. Compared with D5W and D5W plus NO, free water–induced hemolysis led to blunted hemodynamic effects of escalating doses of sodium nitroprusside, which were restored with inhaled NO therapy and oxidation of plasma Hb (P = 0.005 and P = 0.02 for SVRI and CI, respectively). Similar but not statistically significant patterns of response to increasing doses of sodium nitroprusside in the 4 treatment groups were also demonstrated for MAP (C), PAP (D), heart rate, CVP, and PCWP. In fact, all 7 hemodynamic variables demonstrated the expected ordered responses to nitroprusside (P = 0.008 for 7/7 variables having the same response pattern).

Figure 8

Effects of Hb infusions with and without inhaled NO. Infusion of cell-free Hb led to increases in SVRI and pulmonary vascular resistance index that were attenuated by inhaled NO (A and B). In animals breathing air (n = 2), the cell-free Hb remained predominantly oxyhemoglobin (C). In contrast, in animals breathing NO (n = 2), the cell-free Hb was converted to methemoglobin (D).