Nitrite reductase activity of hemoglobin as a systemic nitric oxide generator mechanism to detoxify plasma hemoglobin produced during hemolysis

Abstract

Hemoglobin (Hb) potently inactivates the nitric oxide (NO) radical via a dioxygenation reaction forming nitrate (NO(3)(-)). This inactivation produces endothelial dysfunction during hemolytic conditions and may contribute to the vascular complications of Hb-based blood substitutes. Hb also functions as a nitrite (NO(2)(-)) reductase, converting nitrite into NO as it deoxygenates. We hypothesized that during intravascular hemolysis, nitrite infusions would limit the vasoconstrictive properties of plasma Hb. In a canine model of low- and high-intensity hypotonic intravascular hemolysis, we characterized hemodynamic responses to nitrite infusions. Hemolysis increased systemic and pulmonary arterial pressures and systemic vascular resistance. Hemolysis also inhibited NO-dependent pulmonary and systemic vasodilation by the NO donor sodium nitroprusside. Compared with nitroprusside, nitrite demonstrated unique effects by not only inhibiting hemolysis-associated vasoconstriction but also by potentiating vasodilation at plasma Hb concentrations of <25 muM. We also observed an interaction between plasma Hb levels and nitrite to augment nitroprusside-induced vasodilation of the pulmonary and systemic circulation. This nitrite reductase activity of Hb in vivo was recapitulated in vitro using a mitochondrial NO sensor system. Nitrite infusions may promote NO generation from Hb while maintaining oxygen delivery; this effect could be harnessed to treat hemolytic conditions and to detoxify Hb-based blood substitutes.

Fig. 1.

Cardiovascular effects of nitrite in nonhemolyzing animals. A: serial plasma nitrite levels (in μM) in nonhemolyzing animals. In animals receiving a 6-h infusion of 5% dextrose (D5W), a sodium nitrite infusion of 27.5 mg/h (○) led to a rapid rise and then a sustained plasma nitrite concentration (range: 15–21 μM) compared with a placebo infusion of 0.9% NaCl (•). B–H: in nonhemolyzing animals, sodium nitrite increased the cardiac index (CI; B) and decreased the systemic vascular resistance index (SVRI; C), pulmonary vascular resistance (PVRI; D), mean arterial pressure (MAP; E), mean pulmonary arterial pressure (PAM; F), central venous pressure (CVP; G), and pulmonary artery occlusion pressure (PAOP; H) compared with placebo. Intravenous nitrite enhanced cardiac performance (CI) by arterial vasodilation (SVRI, PVRI, MAP, and PAM) and caused venodilation (CVP).

Fig. 2.

Effects of nitrite on the components of the CI in nonhemolyzing animals. The CI (A) and its components (B) were transformed into the log scale to demonstrate the individual contribution of heart rate (HR) and stroke volume index (SVI) to CI in an additive fashion (normal scale: CI = SVI × HR, log scale: log CI = log SVI + log HR) (68). In animals receiving D5W and nitrite, the nitrite-induced increase in the CI is mediated predominantly through an increase in SVI and to a lesser extent by a chronotropic effect. Over time, the decrease in HR causes further increases in SVI by increasing diastolic filling time in the ventricles, leading to higher end-diastolic volumes. Furthermore, the higher end-diastolic volumes translate into higher end-diastolic pressures, which may explain the increase in PAOP over time (Fig. 1H).

Fig. 3.

Cardiovascular effects of nitrite during intravascular hemolysis. The cardiovascular effects of nitrite (27.5 mg/h) at different levels of cell-free plasma hemoglobin (Hb; 0, < 25 μM, and >25 μM) are shown in A–G. For each parameter, the isolated effect of nitrite is displayed after controlling for animal variability and the independent effects of hemolysis. The depicted value represents the mean change in the parameter from time 0 to 1.5, 3, 4.5, and 6 h for all animals within the specified hemolysis group (x-axis: 0 μM, •; <25 μM, ○; and >25 μM, ▾). According to previous experiments, if nitrite functioned purely as a nitric oxide (NO) donor, then there should be progressive attenuation of the vasodilatory effects of nitrite with increasing levels of hemolysis; the NO generated from nitrite should be progressively scavenged by the increasing levels of cell-free plasma Hb (52). In these experiments, the effect of nitrite was dependent on the level of intravascular hemolysis (P = 0.01 for a differing effect of nitrite at low-level hemolysis compared with zero and high-level hemolysis across the 7 physiologic variables combined). A consistent U-shaped relationship between the physiological effects of nitrite and the levels of cell-free plasma Hb was detected. At low levels of hemolysis (Hb concentration < 25 μM), the vasodilatory effects of nitrite were potentiated, whereas with higher levels of hemolysis (cell-free plasma Hb > 25 μM), the expected inhibition of the vasodilatory effects of nitrite were observed.

Fig. 4.

Nitrite levels and plasma Hb composition during intravascular hemolysis. A and D: intravascular hemolysis occurred at varying rates in animals receiving water and nitrite infusions. In the low-level hemolysis group (Hb < 25 μM; A), the average peak cell-free plasma Hb level was 20 μM; in the high-level hemolysis group (Hb > 25 μM; D), the average peak cell-free plasma Hb level was 142 μM. Animals receiving D5W and nitrite represent the zero-hemolysis control group with all measured cell-free plasma Hb levels <5 μM (data not shown). B and E: total plasma Hb composition in the low-level (B) and high-level (E) hemolysis groups (○, met-Hb; •, oxy-Hb). In animals receiving D5W and nitrite (zero hemolysis), 81% of the measured cell-free plasma Hb was oxy-Hb (depicted as dark gray reference lines) and 19% was met-Hb (depicted as light gray reference lines). With increasing Hb concentrations, the rate of met-Hb formation increased from 0 to 3 h (P = 0.0001), producing higher levels of met-Hb from 3 to 6 h (P = 0.0001) in animals with higher levels of hemolysis compared with animals with lower levels of hemolysis. These results can be explained by the fact that the overall reactions of nitrite with oxy- and deoxy-Hb are second order during their lag phases such that increasing Hb concentrations lead to increasing rates of reaction. C and F: in both the low-level (C) and high-level (F) hemolysis groups, plasma nitrite levels were similar and were maintained within a range of 16–21 μM throughout the 6-h experiment.

Fig. 5.

Effects of nitrite and intravascular hemolysis on cardiovascular responses to sodium nitroprusside. The physiological effects of sodium nitroprusside (a direct NO donor) were dependent on the level (or dose) of hemolysis and the presence of sodium nitrite (P = 0.09, 0.05, and 0.009 for the interaction between level of hemolysis and nitrite on the effect of sodium nitroprusside for CI, SVRI, and PVRI, respectively). Depicted values represent the mean percent changes in the parameter for all doses of nitroprusside for all animals within the specified hemolysis group (0 μM, •; <25 μM, ○, and >25 μM, ▾). As expected for a direct NO donor, in animals not receiving nitrite, sodium nitroprusside-induced increases in CI and decreases in SVRI and PVRI were progressively inhibited by increasing levels of hemolysis (A–C; no nitrite). Compared with nonhemolyzing animals not receiving nitrite (zero hemolysis, no nitrite), nonhemolyzing animals receiving nitrite (zero hemolysis, nitrite) demonstrated blunted effects of sodium nitroprusside on CI, SVRI, and PVRI, suggesting a decreased vasodilator effect of donated NO in the presence of nitrite (A–C). In animals receiving nitrite, the effects of sodium nitroprusside on CI, SVRI, and PVRI were accentuated with low levels of hemolysis (Hb < 25 μM, nitrite) and attenuated with high levels of hemolysis (Hb > 25 μM, nitrite) compared nonhemolyzing animals (zero hemolysis, nitrite) (A–C; nitrite). These findings may be explained by the nitrite reduction reaction with Hb (detailed in results).

Fig. 6.

Nitrite- and Hb-dependent inhibition of mitochondrial respiration shows a U-shaped curve. A: mitochondria (2 mg/ml) were stimulated to respire in the presence of no treatment (solid trace), nitrite (18 μM, dark gray trace), oxygenated Hb (20 μM, mid-gray trace), or nitrite (18 μM) + Hb (20 μM) (light gray trace). Removal of the lid from the sealed chamber is denoted by the arrow. Time to inhibition was measured from removal of the lid to time the trace deviated from 0% oxygen. In these experiments, the oxygen trace deviates from zero once the mitochondria stop respiring due to the exhaustion of substrate or inhibition by NO produced by reactions of Hb with nitrite. B: quantification of several traces similar to those shown in A with different levels of Hb. Inhibition of mitochondrial respiration (secondary to NO production) occurred most rapidly with nitrite and low levels of Hb. The time to inhibition appears to be dependent on the rate of NO production from reactions of nitrite with deoxy-Hb and the rate of NO consumption by excess oxy-Hb. All data are means ± SE of at least 3 independent experiments. *P < 0.01 compared with nitrite alone.