Nitrite infusions induce regional vasodilation in patients with SCD: Baseline blood flow measurements were performed in each patient prior to the infusion of drug. The baseline forearm blood flow in our 14 patients was 5.36 +/- 0.49 ml/min/100 ml of forearm tissue (mean SEM). Forearm blood flow in patients with SCD increased to 5.65 +/- 0.43, 6.50 +/- 0.58 and 9.04 +/- 0.53 ml/min/100 ml of forearm tissue with infusions of 0.4, 4 and 40 micromol/min respectively (P <00001, anova with repeated measures). These values yielded calculated nitrite-induced increase in blood flow over baseline by 7.9 +/- 4.1, 25.1 +/- 7.1 and 77.4 +/- 11.2% respectively following infusions of 0.4, 4 and 40 micromol/min infusions of sodium nitrite (P <00001, anova). Vascular responses were not significantly different in patients on hydroxycarbamide compared to those who were not. These data indicate that sodium nitrite infusions produce significant vasodilation in the regional circulation of the forearm in patients with SCD, the prospectively defined primary hypothesis in this study. Plasma nitrite levels in patients with SCD: The mean baseline plasma nitrite concentration sampled from the intravenous catheter in the antecubital vein of the infused arm was 0.17 +/- 0.02 micromol/l. The mean plasma nitrite concentrations following the 0.4, 4 and 40 micromol/min infusions were 2.4 +/- 0.36, 16.9 +/- 3.6 and 132.2 +/- 23 micromol/l respectively (P <00001, anova). The systemic mean plasma nitrite level 5 min after the infusion of the maximal dose of sodium nitrite was 9.7 +/- 3.5 micromol/l. The venous mean plasma nitrite level in the infused arm 30 min after the maximum sodium nitrite infusion was 5.3 +/- 0.9 micromol/l. The individual regional plasma nitrite levels correlated with the dose of sodium nitrite administered (Spearman correlation r = 0.93, P <00001). Forearm blood flow is related to plasma nitrite: Forearm blood flow in patients with SCD, expressed as percentage increase over baseline, correlated positively with the infused dose of sodium nitrite dose in micromol/kg (Spearman correlation r = 0.70, P <0.0001. Absolute forearm blood flow, expressed as ml/min/100 ml of forearm tissue, correlated positively with plasma nitrite levels achieved with the infusions (Spearman correlation r = 0.51, P = 0.0003. This relationship suggests that sodium nitrite infusions induced vasodilation in patients with SCD in a concentration-dependent fashion. There was no statistically significant correlation between baseline plasma nitrite concentration and baseline forearm blood flow. Blunted nitrite response compared with controls: Vasodilatory responses in patients with SCD were blunted compared to control subjects, even though the sickle cell patients received a 10% higher nitrite dose. Mean arterial pressures did not change significantly after nitrite infusion, which suggests that systemic nitrite administration at these doses does not cause hypotension in patients with SCD. This systemic blood pressure response was blunted compared to healthy controls. The nitrite levels in patients with SCD were comparable to healthy controls at baseline (0.16 +/- 0.02 micromol/l vs. 0.18 +/- 0.17 micromol/l, P = 0.7) and after low dose nitrite infusions (2.4 +/- 0.4 vs. 2.6 +/- 0.5, P = 0.8). The patients with SCD demonstrated somewhat lower regional (132 +/- 23 micromol/l vs. 221 +/- 58 micromol/l, P = 02) and systemic (5.3 +/- 0.9 micromol/l vs. 16 micromol/l) plasma nitrite levels after the highest dose of sodium nitrite infused, but none of these differences were statistically significant. SNP responsiveness in patients with SCD: Consistent with our previously published data (Gladwin et al, 2003), forearm blood flow increase during infusions of the exogenous NO donor, SNP, in patients with SCD was blunted in comparison to 10 African American healthy control subjects. In the patients with SCD, 30 min after the nitrite infusion, the level of forearm blood flow rose globally higher during infusion of SNP at baseline (0), 0.8, 1.6 and 3.2 microg/min, compared to the SNP responses prior to nitrite treatment (prenitrite 5.18 +/- 0.48, 6.45 +/- 0.60, 7.25 +/- 0.48, 8.37 +/- 0.44 ml/min/100 ml vs. postnitrite 5.86 +/- 0.59, 7.40 +/- 0.69, 8.70 +/- 0.77, 9.69 +/- 0.77 ml/min/100 ml, respectively, P <00001, anova with repeated measures). However, nitrite infusion did not improve the SNP responsiveness, as indicated by percentage increase in forearm blood flow. The postnitrite global increase in forearm blood flow remained, but was smaller and no longer statistically significant by approximately 90 min after the nitrite administration, as seen duringinfusions of the NO synthase inhibitor L-NMMA at 0, 4 and 8 micromol/min, compared to the prenitrite L-NMMA infusions (prenitrite 5.57 +/- 0.52, 4.32 +/- 0.45, 4.26 +/- 0.39 ml/min/100 ml vs. postnitrite 5.75 +/- 0.55, 4.70 +/- 0.42, 4.62 +/- 0.50 ml/min/100 ml, respectively, n = 13, P = 02, anova with repeated measures). The failure of NO synthase blockade to eliminate this small trend toward globally increased blood flow suggests that the nitrite-induced increase in blood flow is NOS-independent, but this is not conclusive. At this 90-min time point, the percentage of blood flow that was NO synthase-dependent was not changed significantly by nitrite, as assessed by L-NMMA infusion at 4 micromol/min (-21 +/- 5% vs. -17 +/- 4%), and 8 micromol/min (-21 +/- 4% vs. -20 +/- 3%). These results suggest that nitrite globally increases regional blood flow, but it does not specifically decrease resistance to either exogenous or endogenous NO. The vasodilatory response to the maximal dose of SNP before the nitrite infusion correlated strongly with the response to the maximal dose of nitrite (Spearman r = 075, P = 0002). This indicates that those patients whose vascular response demonstrates resistance to SNP manifest proportional resistance to sodium nitrite. This correlation is consistent with a model in which sodium nitrite, like SNP, functions as an NO donor. Toxicity assessment: The mean prenitrite methaemoglobin level was 1.2 +/- 0.16%. The regional venous methaemoglobin level was not significantly affected by the lowest dose of nitrite (1.3 +/- 0.16%), although small but statistically significant increases from baseline were induced by the infusion of sodium nitrite at 4 micromol/min (1.7 +/- 0.21%, P <005). Regional methaemoglobin levels rose further at 40 micromol/min (4.0 +/- 0.40%, P <0001), which was significantly higher than control subjects at a comparable dose (0.2%, P <0.0001). The systemic methaemoglobin level, measured from venous blood drawn from the contralateral arm, 5 min after infusion of the maximal sodium nitrite dose was 1.8 +/- 0.25%, trending slightly higher than the initial baseline level (P = 006). The regional methaemoglobin level in the infused arm 30 min later was also slightly above baseline, (1.95 +/- 0.2%, P = 001). None of the patients demonstrated clinical signs or symptoms related to elevated methaemoglobin levels (30 - 50%), such as cyanosis or shortness of breath. The venous plasma nitrite levels correlated positively with the venous methaemoglobin level, sampled at the corresponding time point in the study (r = 0.62, P <0.0001). This indicates that blood methaemoglobin levels are a reasonable proxy for plasma nitrite levels. One patient reported transient nausea at the highest dose of nitrite, with no other apparent ill effects. No other symptoms were reported by the patients.
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