Inhaled nitric oxide enables artificial blood transfusion without hypertension

Abstract

Background: One of the major obstacles hindering the clinical development of a cell-free, hemoglobin-based oxygen carrier (HBOC) is systemic vasoconstriction.

Methods and results: Experiments were performed in healthy mice and lambs by infusion of either murine tetrameric hemoglobin (0.48 g/kg) or glutaraldehyde-polymerized bovine hemoglobin (HBOC-201, 1.44 g/kg). We observed that intravenous infusion of either murine tetrameric hemoglobin or HBOC-201 induced prolonged systemic vasoconstriction in wild-type mice but not in mice congenitally deficient in endothelial nitric oxide (NO) synthase (NOS3). Treatment of wild-type mice by breathing NO at 80 ppm in air for 15 or 60 minutes or with 200 ppm NO for 7 minutes prevented the systemic hypertension induced by subsequent intravenous administration of murine tetrameric hemoglobin or HBOC-201 and did not result in conversion of plasma hemoglobin to methemoglobin. Intravenous administration of sodium nitrite (48 nmol) 5 minutes before infusion of murine tetrameric hemoglobin also prevented the development of systemic hypertension. In awake lambs, breathing NO at 80 ppm for 1 hour prevented the systemic hypertension caused by subsequent infusion of HBOC-201.

Conclusions: These findings demonstrate that HBOC can cause systemic vasoconstriction by scavenging NO produced by NOS3. Moreover, in 2 species, inhaled NO administered before the intravenous infusion of HBOC can prevent systemic vasoconstriction without causing methemoglobinemia.

Figure 1

(A) In WT mice, tail-cuff systolic blood pressure (SBP, mmHg) was measured before and after IV infusion of whole blood (n=7), murine tetrameric Hb (n=5), or HBOC-201 (n=5). *p<0.001 differs versus whole blood infusion group. (B) In NOS3^−/− mice, SBP was measured before and after IV infusion of whole blood (n=5), murine tetrameric Hb (n=5), or HBOC-201 (n=5). There was no increase of SBP after infusion of murine tetrameric Hb or HBOC-201.

Figure 2

(A) Tail-cuff SBP (mmHg) was measured in awake WT mice after infusion of murine tetrameric Hb solution while either breathing air or breathing 80 ppm NO in air (iNO) (n=5 in each group). *p=0.004 differs versus group breathing air without NO. (B) Tail-cuff SBP (mmHg) was measured before and after IV infusion of murine tetrameric Hb containing various concentrations of metHb (3%, 6%, 14.5%, and 100%, respectively n=5 for each group). *p<0.001 differs versus before infusion.

Figure 3

(A) Tail-cuff SBP (mmHg) was measured after pretreatment by breathing 80 ppm NO for 1 h and subsequent infusion of murine tetrameric Hb solution (n=5). Additional mice received the murine tetrameric Hb solution without NO pretreatment (n=7). (B) Plasma metHb concentration (%) at various times after infusion of murine tetrameric Hb following pretreatment by breathing 80 ppm NO in air for 1 h (n=5). (C) SBP was measured after infusion of murine tetrameric Hb solution in mice pretreated without or with breathing 80 ppm NO for 15 min (n=5 in each group). (D) SBP was measured after infusion of HBOC-201 in mice pretreated without or with breathing 80 ppm NO for 15 min (n=5 in each group). (E) SBP was measured after infusion of murine tetrameric Hb solution in mice pretreated without or with breathing 200 ppm NO for 7 min (n=5 in each group). *p<0.05 differs versus group breathing air without NO.

Figure 4

(A) Tail-cuff SBP (mmHg) was measured in awake mice before and after infusion of whole blood (n=7), murine tetrameric Hb (n=5), or nitrite (48 nmol) followed by murine tetrameric Hb (n=5). *p<0.05 differs versus both the whole blood group and the nitrite plus murine tetrameric Hb group. (B) MetHb concentration (%) in whole blood and plasma (n=5) at baseline and 10 min after infusion of nitrite. *p<0.05 differs versus baseline plasma level.

Figure 5

(A) Mean arterial pressure (MAP, mmHg) of awake lambs after infusion of autologous whole blood while breathing at FiO₂=0.3 (n=3), HBOC-201 (n=3), or HBOC-201 after pretreatment with breathing 80 ppm NO for 1 h (n=5). * p<0.05 differs from autologous blood and from HBOC-201 after inhaled NO. (B) Plasma metHb concentration (%) before and after infusion of either autologous whole blood or HBOC-201 with or without pretreatment by inhaled NO. *p<0.05 autologous whole blood differs from HBOC-201 with or without iNO pretreatment. Pretreatment with inhaled NO did not increase plasma metHb.