Red cell survival and NO scavenging in sickle cell disease

Nitric oxide (NO), a ubiquitous and versatile signaling molecule, reacts readily with both oxy- and deoxyhemoglobin. During the first decade of this century, multiple experimental and clinical studies along with a number of review articles were published based on the hypothesis that NO depletion by elevated levels of plasma hemoglobin in the microcirculation plays a central role in the pathogenesis of sickle cell disease (SCD) 1-5. These papers concluded that those patients with a relatively increased degree of hemolysis are at higher risk of developing pulmonary hypertension, leg ulcers, priapism, and perhaps stroke. The vast bulk of the data supporting these claims was based on indirect assessments of hemolysis by means of reticulocyte counts and measurements of serum LDH and bilirubin. The paper by Quinn et al. 6 that appears in this issue of the American Journal of Hematology provides critically important new information on red cell survival measurements in sickle cell patients that pertain directly to the role of hemolysis in the clinical manifestations of this disease. State of the art methodology involving the oral administration of a bolus of stable 15N-glycine followed by highly precise measurements of the mole fraction of this isotope in heme within circulating red cells has enabled the construction of highly accurate red cell survival curves. In some patients, a bi-phasic pattern provides a clear depiction of the contribution of the subpopulation of RBC that has relatively prolonged survival owing to the presence of Hb F. Although only 11 SS patients were studied, the data are so robust that firm and highly relevant conclusions can be drawn. The authors found that RBC survival was inversely correlated with reticulocyte count (P < 0.01) but was not significantly correlated with serum LDH, AST, and either total or non-conjugated bilirubin. Moreover measurements of tricuspid jet regurgitant velocity, a surrogate marker of pulmonary hypertension, correlated directly with serum LDH but not with red cell survival. The level of plasma hemoglobin is clearly the index of hemolysis most relevant to the proposal that NO scavenging by hemoglobin is responsible for certain complications of SCD. Clinical symptoms directly attributable to NO scavenging have been convincingly documented in patients with high levels of plasma hemoglobin. For example, about two thirds of those with paroxysmal nocturnal hemoglobinuria (PNH) have esophageal spasm with tightness of the chest and difficulty swallowing, particularly early in the morning and during hemolytic episodes. This symptom is completely eliminated by eculizumab, which causes a reduction in plasma hemoglobin to near normal levels 7. As in PNH, NO scavenging is responsible for disordered GI motility encountered in patients receiving intravenous solutions of hemoglobin-based blood substitutes 8. These gastro-intestinal symptoms are not encountered in patients with SCD. In striking contrast to patients with PNH, plasma hemoglobin levels are only modestly elevated in those with SCD. With optimal handling and methodology a mean of 8 mg/dL has been found in patients not in acute crisis, compared to a range in normals of 0.1–1.2 mg/dL 9. More recent data on plasma hemoglobin levels are shown in Fig. 1. Both the measurements reported by Reiter et al. in the left-hand panel and those, on the right, taken from Fig. 3E of the current paper by Quinn et al. confirm earlier reports that the great majority of sickle cell patients have only modest elevations, consistent with extra-vascular hemolysis. However both panels in Fig. 1 show a minority of patients with clearly elevated values. Do these patients have “hyperhemolysis,” and if so, what is the probable cause? Although increased sensitivity to complement and enhanced susceptibility to mechanical stress have been invoked as contributors to hemolysis in SCD 10, clinical documentation has been elusive. Sustained immune hemolysis is an uncommon complication of SCD. It is far more likely that these outliers are false elevations owing to in vitro hemolysis during or following the withdrawal of venous blood specimens 11. Sickle cell patients commonly have difficult venous access and therefore there is increased probability that in vitro hemolysis will occur during blood drawing. In support of the notion that these elevated values are artifacts of improper sample collection, Quinn et al. found no correlation of plasma hemoglobin with their measurements of red cell life span. Left: Levels of plasma free hemoglobin in SS patients and normal individuals (AA). From Reiter et al. 1. Right: Lack of relationship between plasma free hemoglobin and red cell survival in SS patients. From Figure 3E, Quinn et al. 6. [Color figure can be viewed at wileyonlinelibrary.com] These elegant, accurate, and highly relevant determinations of red cell survival in SCD by Quinn et al. offer definitive support for the concluding prescient statement in the 1971 report by Naumann et al. 9: “Hyperhemolysis” in sickle cell (disease) is minor, presumably secondary, and different in principle from the massive intravascular hemolysis observed in immunohemolytic anemias, hereditary enzyme deficiencies or hemolytic septicemias.”