Sensitivity and specificity of I₃⁻/ozone-based chemiluminescence assay, with and without KCN/K₃Fe(CN)₆, for the quantification of total nitrosylated hemoglobin, SNO-Hb, and Hb(FeII)NO. A demonstrates the reaction of standards of pure SNO-Hb (stippled bars) and Hb(FeII)NO (slashed bar) with and without KCN and K₃Fe(CN)₆ (indicated as + and −KCN, respectively), which selectively removes the NO from heme while preserving the S-nitrosothiol bond. Within 5 min, treatment with a large molar excess of KCN/K₃Fe(CN)₆ (0.2 M) eliminates 90% of the signal of Hb(FeII)NO in the chemiluminescent I₃⁻ reaction, while preserving the signal from SNO-Hb (n = 5). Results are expressed as the percentage of the original signal from the standards reacted without KCN/K₃Fe(CN)₆ pretreatment. Similar results were observed up to 120 min (data not shown). B demonstrates the NO signal released from three injections of water from the Sephadex G25 column (background control), followed by increasing standard dilutions of SNO-Hb, pretreated with KCN/K₃Fe(CN)₆ (control hemoglobin, 0.002%, 0.004%, 0.008%, 0.016%, 0.032%), measured by I₃⁻/ozone-based chemiluminescence. C demonstrates measurement linearity of four serial dilutions of SNO-Hb, pretreated with KCN/K₃Fe(CN)₆, from 0.002% to 0.032% (r² = 0.996; P < 0.001; n = 4). Similar results were obtained for standard curves of Hb(FeII)NO (r² = 0.999, P < 0.001, n = 5).

Plasma nitrate and nitrite and FeIII levels at baseline and during 80 ppm NO breathing. A shows venous plasma nitrate (solid line) and whole blood FeIII (dashed line) levels measured for 2 h before NO inhalation, for 2 h during 80 ppm NO inhalation (x axis bar denotes NO inhalation), and for 2 h after NO inhalation. A significant increase, compared with baseline levels, in both plasma nitrate and erythrocyte FeIII is observed at 1 and 2 h of NO inhalation (*, P < 0.01 for both 1- and 2-h NO inhalation time points; n = 7). B shows arterial (solid bars) and venous (stippled bars) plasma nitrite levels from seven individuals at baseline and during 80 ppm NO inhalation. There is no significant increase in nitrite during NO breathing. P values are reported for arterial–venous differences.

Baseline and augmented levels of total nitrosylated hemoglobin (NO on either the heme or cysteine 93) in the arterial and venous circulation. This figure represents the means and standard errors for total nitrosylated hemoglobin in the arterial (solid line) and venous (dashed line) blood from five individuals before and during (solid bar on x axis) 80 ppm NO breathing. A trend toward an arterial venous gradient before (P = 0.09) and after (P = 0.11) NO breathing is observed. During NO breathing, a significant increase occurs in both the levels of arterial and venous nitrosylated hemoglobin (P < 0.001) and the arterial–venous gradient (P = 0.002).

Basal and augmented levels of Hb(FeII)NO or SNO-Hb in eight normal volunteers measured with I₃⁻/ozone-based chemiluminescence with and without KCN and K₃Fe(CN)₆ pretreatment, which selectively removes the NO from heme without affecting NO covalently bound to cysteine 93. A demonstrates the effects of KCN and K₃Fe(CN)₆ pretreatment on the signal of NO released from hemoglobin in I₃⁻ from a normal individual breathing 80 ppm NO for 2 h. KCN/K₃Fe(CN)₆ pretreatment is indicated by a + sign and no pretreatment by a − sign. A significant reduction in the NO signal after KCN and K₃Fe(CN)₆ pretreatment provides evidence that the majority of the NO associated with hemoglobin is bound to the heme group [Hb(FeII)NO]. The residual signal is still greater than baseline, suggesting that SNO-Hb does form at lower levels. Arterial (A)–venous (V) gradients are observed. B shows the means and standard errors for Hb(FeII)NO and SNO-Hb in the arterial (solid bars) and venous blood (stippled bars) from eight individuals at baseline and during 80 ppm NO breathing. Arterial-venous gradients in Hb(FeII)NO are significant (P = 0.019), whereas those of SNO-Hb are not (P = 0.55).