NATURE MEDICINE • VOLUME 8 • NUMBER 12 • DECEMBER 2002 1383 ARTICLES Nitric oxide (NO) reacts at nearly diffusion-limited rates with oxyhemoglobin and deoxyhemoglobin to produce nitrate plus methemoglobin 1,2 and iron-nitrosylhemoglobin 3,4 , respectively; however, the rate of NO scavenging is reduced 1,000-fold by se- questering hemoglobin within the red cell membrane 5,6 . The rates of NO consumption by cell-free and intraerythrocytic he- moglobin suggest that only when hemoglobin is physically compartmentalized within erythrocytes will NO produced by endothelial cells reach concentrations within smooth muscle necessary to activate guanylyl cyclase and cause vasodilation 6–8 . Such an effect is believed to be the cause of pulmonary and sys- temic hypertension 9–11 , decreased organ perfusion 12,13 , esophageal (smooth muscle) spasm 14 and increased mortality 15 , which occur after infusions of stroma-free hemoglobin into ani- mals or humans as an oxygen-carrying, artificial blood substi- tute. In contrast, heme-pocket mutations that reduce hemoglobin–NO affinity ameliorate the hypertensive effect of cell-free hemoglobin 16 . Several diffusional factors contribute to the negative modula- tion of the reaction of NO with ferrous hemoglobin and include the erythrocyte membrane, or sub-membrane network of pro- tein 17,18 , an unstirred layer surrounding the erythrocyte 6,19 , and an erythrocyte-free zone within the lumen nearest to the en- dothelial layer that results from dynamic, flow-mediated, axial concentration of the red cells 8,20,21 . Decompartmentalized, cell- free hemoglobin is not constrained by these physical factors, and as little as 6 μM cell-free oxyhemoglobin eliminates NO- mediated basal vasodilation 22 . In addition, hemoglobin dissoci- ates into dimers when released into plasma. These smaller species extravasate from the vascular lumen to positions be- tween endothelial cells and smooth muscle, and may magnify NO scavenging. We therefore hypothesized that sickle-cell disease, character- ized by high concentrations of decompartmentalized hemoglo- bin secondary to acute and chronic hemolysis, would limit NO bioavailability as a direct result of cell-free hemoglobin- mediated NO dioxygenation and nitrosylation. Intravascular hemolysis in sickle-cell disease Polymerization of hemoglobin S in patients with sickle-cell dis- ease leads to the destabilization of the red blood cell membrane and the premature destruction of 10% of the total number of erythrocytes every 24 hours, which is equivalent to decompart- mentalization of up to 30 g of hemoglobin per day. Approximately 30% of the total hemolysis occurs intravascu- larly 23 and is sufficient to saturate the hemoglobin scavenging system. As expected, laboratory data from 27 patients with sickle-cell disease in steady state (not in acute pain crisis) reveal significant evidence of hemolysis including elevated levels of bilirubin and lactate dehydrogenase, and an accompanying profound anemia and reticulocytosis (Table 1). Plasma hemo- globin levels (Fig. 1a) and heme concentrations (4.2 ± 1.1 μM; Fig. 1b) were elevated, and haptoglobin was undetectable in 21 of 23 patients assayed (Fig. 1a and Table 1), indicating satura- Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease CHRISTOPHER D. REITER 1,2 , XUNDE WANG 1 , JOSE E. TANUS-SANTOS 1,2 , NEIL HOGG 3 , RICHARD O. CANNON III 4 , ALAN N. SCHECHTER 2 & MARK T. GLADWIN 1,2 1 Critical Care Medicine Department of the Warren G. Magnuson Clinical Center, 2 Laboratory of Chemical Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA 3 Biophysics Research Institute and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA 4 Cardiovascular Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA Correspondence should be addressed to M.T.G.; e-mail: mgladwin@nih.gov Published online 11 November 2002; doi:10.1038/nm799 Although the deleterious vasoconstrictive effects of cell-free, hemoglobin-based blood substi- tutes have been appreciated, the systemic effects of chronic hemolysis on nitric oxide bioavail- ability have not been considered or quantified. Central to this investigation is the understanding that nitric oxide reacts at least 1,000 times more rapidly with free hemoglobin solutions than with erythrocytes. We hypothesized that decompartmentalization of hemoglobin into plasma would divert nitric oxide from homeostatic vascular function. We demonstrate here that plasma from patients with sickle-cell disease contains cell-free ferrous hemoglobin, which stoichiomet- rically consumes micromolar quantities of nitric oxide and abrogates forearm blood flow re- sponses to nitric oxide donor infusions. Therapies that inactivate plasma hemoglobin by oxidation or nitric oxide ligation restore nitric oxide bioavailability. Decompartmentalization of hemoglobin and subsequent dioxygenation of nitric oxide may explain the vascular complica- tions shared by acute and chronic hemolytic disorders. © 2002 Nature Publishing Group http://www.nature.com/naturemedicine