Inhaled nitric oxide augments nitric oxide transport on sickle cell hemoglobin without affecting oxygen affinity

Abstract

Nitric oxide (NO) inhalation has been reported to increase the oxygen affinity of sickle cell erythrocytes. Also, proposed allosteric mechanisms for hemoglobin, based on S-nitrosation of beta-chain cysteine 93, raise the possibility of altering the pathophysiology of sickle cell disease by inhibiting polymerization or by increasing NO delivery to the tissue. We studied the effects of a 2-hour treatment, using varying concentrations of inhaled NO. Oxygen affinity, as measured by P(50), did not respond to inhaled NO, either in controls or in individuals with sickle cell disease. At baseline, the arterial and venous levels of nitrosylated hemoglobin were not significantly different, but NO inhalation led to a dose-dependent increase in mean nitrosylated hemoglobin, and at the highest dosage, a significant arterial-venous difference emerged. The levels of nitrosylated hemoglobin are too low to affect overall hemoglobin oxygen affinity, but augmented NO transport to the microvasculature seems a promising strategy for improving microvascular perfusion.

Figure 1

Effect of inhaled NO on arterial and venous pO₂ in SS and AA individuals. (a) Before NO inhalation (hours 1 and 2), the SS arterial pO₂ is lower than the AA arterial pO₂. The SS venous pO₂ values are similar to the AA venous pO₂ values, reflecting the reduced oxygen extraction or arterialization of venous blood that is characteristic of patients with sickle cell anemia. During 2 hours of 80 ppm NO inhalation (hours 3 and 4; filled horizontal bar), there is no significant change in AA or SS pO₂ for arterial and venous blood. (b) During dose escalation of inhaled NO in 3 SS individuals, there is no significant change in arterial or venous pO₂ during 1-hour inhalation of 40, 60, and 80 ppm NO gas.

Figure 2

Effect of inhaled 80 ppm inhaled NO on plasma NO_X (nitrate and nitrite measured by the Griess reaction) and methemoglobin. (a) For 2 hours before NO inhalation, mean plasma levels of NO_X in SS and AA individuals were 10 ± 8 μM and 12 ± 15 μM, respectively. After 2 hours of inhalation (hours 3 and 4; filled horizontal bar), mean levels rose significantly (P < 0.001) to 85 ± 8 μM in the SS individuals and 60 ± 15 μM in the AA individuals. After NO inhalation, there was a steady decrease in plasma NO_X concentration. (b) During NO inhalation (hours 3 and 4; filled horizontal bar), mean methemoglobin levels rose from 0.3 ± 0.0% in the SS individuals and 0.24 ± 0.02% in the AA individuals to 1.2 ± 0.4% and 0.9 ± 0.09%, respectively (P = 0.001). After correcting for hemoglobin concentration, there was no difference in absolute methemoglobin values between AA and SS individuals. Values returned to baseline within 1–2 hours in all individuals.

Figure 3

Effect of inhaled NO on the oxygen affinity (P₅₀) of SS and AA erythrocytes. (a) The AA ODCs (expressed as P₅₀) at baseline before NO inhalation (hours 1 and 2) demonstrated minimal hysteresis when measured during sample deoxygenation or during sample oxygenation. The baseline SS ODCs measured at 1 and 2 hours before NO inhalation, as expected, demonstrated significant hysteresis during sample deoxygenation and during sample oxygenation. There was no significant change in any of the P₅₀ measurements after 1 and 2 hours of inhaled NO at 80 ppm in either the AA or SS individuals (hours 3 and 4; filled horizontal bar), measured either during sample deoxygenation (for SS individuals; P = 0.63) or reoxygenation (for SS individuals; P = 1.00). There was no significant change in any of the P₅₀ measurements for up to 3 hours after NO inhalation in any of the individuals tested (10/10 tested in the first hour after NO; 7/10 in the second hour; and 3/10 in the third hour). (b) During dose escalation of inhaled NO in 3 SS individuals, there is no significant change in P₅₀ measured during sample deoxygenation or reoxygenation after 1 hour inhalation each of 40, 60, and 80 ppm NO gas.

Figure 4

HPLC-electrospray ionization mass spectrometry of SNO-hemoglobin digested with endoproteinase Glu-C confirms specificity of S-nitrosocysteine reaction with hemoglobin β-chain cysteine 93. (a) Shown is the characteristic chromatogram, after enzymatic digestion of hemoglobin, of the 11–amino acid fragment (hemoglobin β-chain amino acids 91–101) that includes cysteine 93. The molecular weight is 1,305.5, with 3 predominant charge states (+4, +3, +2) with respective m/z of 327.4, 436.2, and 653.7 (inset). (b) Digestion of SNO-hemoglobin results in an increased retention time on the column and an increase in the molecular weight of the 11–amino acid fragment to 1,334.5, consistent with the addition of 29 mass units (NO has 30 mass units minus 1 mass unit of hydrogen lost during covalent binding). The m/z values of the 3 charge states (+4, +3, +2) increase to 334.6, 445.8, and 668.2, respectively (inset).

Figure 5

Chemiluminescent detection of NO released from hemoglobin reacted in I₃^– before and after dose escalation of inhaled NO. NO signal is measured in millivolts (y-axis), and time is measured in seconds (x-axis). (a) Sample measurements before NO inhalation are shown. The first 2 peaks are from 200 μL of water collected from the G25 column immediately before use. The following 4 peaks are from 200 μL of lysed desalted hemoglobin from the venous (v) and arterial (a) blood injected in duplicate in alternating sequence. An arterial-venous difference is not appreciated, and the signal is very low (0.0035% moles NO per moles heme subunit; calculated by dividing the area under the curve [after subtraction from G25 water] from each injection by the concentration of hemoglobin in the sample, multiplied by 100). (b) The signal obtained after breathing 40 ppm NO gas for 1 hour. The venous (0.014%) and arterial (0.011%) nitrosylated hemoglobin levels have risen. (c) The signal obtained after breathing 60 ppm NO gas for 1 hour exhibits a difference between the arterial (0.02%) nitrosylated hemoglobin level and the venous (0.014%), although this difference was not observed in all patients studied. (d) Inhalation of 80 ppm NO further increased the arterial-venous difference (arterial 0.024% and venous 0.017%). This difference was observed in all 3 individuals studied.

Figure 6

Effect of dose escalation of inhaled NO on nitrosylated hemoglobin, expressed as the percentage moles of NO per moles of heme subunits. For 3 hours before NO inhalation, the mean percentage of nitrosylated hemoglobin in the arterial blood was 0.004% and in the venous blood, 0.004%. There is a significant stepwise increase in nitrosylated hemoglobin during NO inhalation in both arterial (0.011% on 40 ppm, 0.016% on 60 ppm, and 0.022% on 80 ppm) and venous (0.014% on 40 ppm, 0.016% on 60 ppm, and 0.016% on 80 ppm) blood (P < 0.05 for all increases except the venous sample at 40 ppm, for which P = 0.075). A significant arterial-venous difference was observed on 80 ppm inhaled NO (P = 0.02).